Solid phase extraction with capillary electrophoresis

ABSTRACT

Methods, systems and devices that provide fluid devices with at least one SPE bed adjacent (upstream of) a separation channel which may be in communication with an inlet of a Mass Spectrometer. The fluid device can be configured to operate using independently applied pressures to a BGE reservoir and a sample reservoir for pressure-driven injection that can inject a discrete sample plug into a separation channel that does not require voltage applied to the sample reservoir and can allow for in-channel focusing methods to be used. The methods, systems and devices are particularly suitable for use with a mass spectrometer but optical or other electronic detectors may also be used with the fluidic devices.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/636,822, filed Jun. 29, 2017, which is a divisional of U.S. patentapplication Ser. No. 15/079,541, filed Mar. 24, 2016, which claims thebenefit of and priority to U.S. Provisional Application Ser. No.62/243,919, filed Oct. 20, 2015, the contents of which are herebyincorporated by reference as if recited in full herein.

FIELD OF THE INVENTION

This invention is related to sample processing that may be particularlysuitable for electrospray ionization and/or sample processing systemsthat interface with mass spectrometers.

BACKGROUND OF THE INVENTION

Electrospray ionization (“ESI”) is an important technique for theanalysis of biological materials contained in solution by massspectrometry. See, e.g., Cole, R. B. Electrospray Ionization MassSpectrometry: Fundamentals, Instrumentation & Applications; John Wileyand Sons, Inc.: New York, 1997. Electrospray ionization was developed inthe late 1980s and was popularized by the work of Fenn. See, e.g., FennJ B, Mann M, Meng C K, Wong S F & Whitehouse C M (1989), Electrosprayionization for mass-spectrometry of large biomolecules, Science 246,64-71. Simplistically, electrospray ionization involves the use ofelectric fields to disperse a sample solution into charged droplets.Through subsequent evaporation of the droplets, analyte ions containedin the droplet are either field emitted from the droplet surface or theions are desolvated resulting in gas phase analyte ions. The source ofthe liquid exposed to the electric field and to be dispersed is ideallyone of small areal extent as the size of the electrospray emitterdirectly influences the size of droplets produced. Smaller dropletsdesolvate more rapidly and have fewer molecules present per dropletleading to greater ionization efficiencies. These ions can becharacterized by a mass analyzer to determine the mass-to-charge ratio.Further analyte structural information can be obtained by employingtandem mass spectrometry techniques.

Separation of analytes prior to electrospray ionization is important forminimizing ionization suppression and MS spectral complexity.Microfluidic capillary electrophoresis with integrated electrosprayionization has been demonstrated as a fast and efficient method ofcoupling a liquid phase chemical separation with mass spectroscopydetection. See, e.g., Anal. Chem. 2008, 50, 6881-6887; and Anal. Chem.2015, 87, 2264-2272. Conventional microfluidic methods that employelectrokinetic flow of sample into the separation channel are subject toinjection bias and cannot effectively be used for some on-device samplefocusing methods. Further, the injection of a well-defined band ofsample into the separation channel of the microfluidic device can beimportant to achieving an efficient separation.

Most of the efforts to integrate sample processing with CE can beclassified as either electrophoretic or chromatographic based.Electrophoretic based techniques, including sample stacking, sweeping,pH induced stacking, and transient isotachophoresis (tITP), can besimple to implement and can require little instrumentation development.Unfortunately, these techniques cannot typically load a sample volumelarger than the volume of the capillary which limits the achievableconcentration and sensitivity improvement. Furthermore, electrophoreticmethods often concentrate matrix components equally to the analytes ofinterest, which can reduce separation performance. Finally, thesemethods can be limited to a narrow scope of analyte and bufferconditions, and may not be as widely applicable as other sampleprocessing techniques.

On the other hand, chromatographic-based techniques, such as solid phaseextraction (SPE), are typically more versatile thanelectrophoretic-based methods and can offer higher pre-concentrationvalues based on the ability to load multiple capillary volumes onto thechromatographic sorbent. See, e.g., Ramautar et al. Electrophoresis2014, 35, 128-137, the contents of which are hereby incorporated byreference herein. However, these methods present their own shortcomings.The presence of the SPE sorbent in the separation capillary can lead toclogging and disruption of the electroosmotic flow (EOF), reducingseparation performance. Furthermore, in this scenario, matrix componentsenter the separation capillary, which can lead to wall interactions andfurther diminish the separation performance. On-line coupling, where theSPE sorbent is separate from the CE capillary but connected via a flowstream with tubing and valves, is the most common method for combiningSPE with CE. The decoupling of the SPE sorbent from the CE capillary caninhibit or prevent clogging and EOF disruption. Additionally, theinclusion of valves between the SPE sorbent and the CE capillary candirect the matrix components to waste and prevent them from entering theCE capillary. Unfortunately, on-line coupling of SPE and CE oftenrequires complex instrumentation. Furthermore, the transfer of thesample band from the SPE sorbent to the CE capillary typicallyintroduces band broadening, limiting the resulting separationperformance. Additionally, dead volume present in the on-line system candilute the concentrated analyte band, reducing the amount ofpre-concentration that can be achieved. Coupling sample processing withCE without sacrificing the separation performance can be a verychallenging task.

SUMMARY OF EMBODIMENTS OF THE INVENTION

Embodiments of the invention are directed to microfluidic sampleprocessing systems and methods that integrate sample processing andemploy on-line solid phase extraction (SPE) with microchip CE-ESI and/ormicrochip CE-detector systems.

Embodiments of the invention provide fluidic systems and methods thatcan integrate sample processing with on-line SPE using fluidic devices.

Embodiments of the invention provide SPE-CE-MS and/or SPE-CE-detectorsystems and methods that can be configured to eliminate or reduce bandbroadening imparted on the sample during the transfer between the SPEbed and CE capillary using transient isotachophoresis (tITP) to refocusan analyte band prior to separation. The tITP can act as a non-linearelectrophoretic focusing technique where the sample is sandwichedbetween a leading fast moving electrolyte (LE) and a trailing slowermoving electrolyte (TE). Sample analytes will focus into discreet zonesbased upon their electrophoretic mobilities. By utilizing thistechnique, band broadening imparted onto the sample during the SPEelution and transfer step can be reduced, resulting in a narrowinjection plug allowing for a high performance separation. Samplepre-concentration factors can be more than two orders of magnitudewithout any reduction in separation performance.

In some particular embodiments, CE-ESI-MS and/or CE-optical detectorsystems can employ fluidic devices with simple, pressure-driveninjection methods that can independently be applied to a plurality ofdifferent fluid reservoirs.

In some embodiments, pressure-driven sample loading and/or injectionmethods can also be used with on-device sample focusing methods such astransient isotachophoresis.

Embodiments of the invention are directed to methods of sampleprocessing. The methods include providing a fluidic device with at leastone microfluidic or nanofluidic separation channel in fluidcommunication with a background electrolyte (BGE) reservoir, and ananofluidic or microfluidic sample channel in fluid communication withthe separation channel. The sample channel includes at least one solidphase extraction (SPE) bed. The method also includes flowing a samplethrough the sample channel, across the at least one SPE bed, and intothe separation channel then electrophoretically separating an analytecomponent from the sample in the separation channel and performing atleast one of: (a) electrospraying the analyte component from at leastone emitter in fluid communication with the separation channel toward atleast one of a collection device or an inlet of a mass spectrometer; and(b) detecting a signal corresponding to the analyte component in, oremerging from, the separation channel.

The method can include pre-concentrating the sample prior to theelectrophoretic separation.

The sample can be an ionized sample.

The pre-concentrating can include flowing the ionized sample in theseparation channel in a first direction following a leading electrolytewith a first mobility, and in advance of a trailing electrolyte with asecond mobility lower than the first mobility, so that if the samplecomprises multiple components, a component of the sample having ahighest mobility flows directly behind the leading electrolyte, and acomponent of the sample having a lowest mobility flows directly inadvance of the trailing electrolyte, in the first direction.

The sample can be flowed across the at least one SPE bed and into theseparation channel without directing the sample through a valve.

The electrophoretically separating the analyte component can includeapplying an electric field to the fluidic device so that at least acomponent of the electric field is parallel to an axial direction of aportion of the separation channel.

The applying the electric field can include applying an electricalpotential difference between a first position in the BGE reservoir and asecond position downstream from the first position. The second positioncan be located in the separation channel or in a pump channel or in areservoir in fluid communication with one or both of the separation orthe pump channel of the fluidic device.

The method can include flowing an ion-pairing agent across the at leastone SPE bed in advance of, or together with, the sample; rinsing the ionpairing agent from the sample channel into a waste channel in fluidcommunication with the sample channel; and after rinsing the ion pairingagent from the sample channel, flowing an elution fluid in the samplechannel before or during the electrophoretic separation.

The ion-pairing agent can include trifluoroacetic acid (TFA).

The method can include, after flowing the sample through the samplechannel and across the at least one SPE bed, flowing an elution fluidthrough the sample channel and across the at least one SPE bed.

The method can include, prior to flowing the sample across the at leastone SPE bed, pre-conditioning the SPE bed by flowing a pre-conditioningfluid across the at least one SPE bed and into a waste reservoir of thefluidic device.

The method can include, prior to flowing the sample through the samplechannel, forming the at least one SPE bed in the sample channel byflowing a SPE material into the sample channel.

The SPE bed has a length, measured along a flow direction defined by thesample channel, that can be between 100 μm and 1000 μm, and a volumethat can be between about 50 pL to about 10 nL.

The method can include using a blocking member to at least partiallyocclude the sample channel. The blocking member can be positionedadjacent to an end of the at least one SPE bed that is closest to theseparation channel.

The method can include flowing the sample through the sample channel bypressurizing sealed headspaces of the BGE reservoir, a waste reservoir,and a sample reservoir comprising the sample. The BGE reservoir, thewaste reservoir, and the sample reservoir can each be in fluidcommunication with the separation channel. The sample can be flowedthrough the sample channel without applying a voltage to the samplereservoir, to the BGE reservoir, or to the waste reservoir and/or withno electric potential gradient in any of the sample channel, the BGEchannel and the waste channel.

The method can include applying hydrodynamic pressure to cause thesample to flow through the sample channel, across the at least one SPEbed, and into the separation channel.

The applying hydrodynamic pressure, where used, can include (a) first,concurrently applying pressures of between 0.1 pounds per square inch(psi) and 50 psi to each of a sealed headspace of a waste reservoir ofthe fluidic device and a sealed headspace of a sample reservoir of thefluidic device. A pressure applied to the sealed headspace of the wastereservoir is less than a pressure applied to the sealed headspace of thesample reservoir. The method then includes: (b) second, concurrentlyapplying pressures to a sealed headspace of the BGE reservoir and to thesealed headspace of the sample reservoir; and (c) third, reducing thepressure applied to the sealed headspace of the sample reservoir andapplying a pressure of between 0.1 psi and 50 psi to the sealedheadspace of the BGE reservoir so that the pressure applied to thesealed headspace of the BGE reservoir is greater than the pressureapplied to the sealed headspace of the sample reservoir to flush thesample from the sample channel into the separation channel.

The electrophoretically separating the analyte component from the samplecan include reducing the pressure applied to the sealed headspace of theBGE reservoir and applying an electrical potential difference between afirst position in the BGE reservoir and a second position downstreamfrom the first position, when the sample is in the separation channel.

The electrophoretically separating the analyte component from the samplecan include removing a pressure applied to a sealed headspace of the BGEreservoir while applying and electrical potential difference between afirst location in the BGE reservoir and a second position downstreamfrom the first position.

The second/(b) step can be carried out so that the concurrent pressuresapplied to the sealed headspaces of the BGE reservoir and the samplereservoir are each between 0.5 psi and 50 psi, and the third/(c) stepcan be carried out so that no pressure is applied to the sealedheadspace of the sample reservoir and the pressure applied to the sealedheadspace of the BGE reservoir to flush the sample is between 0.1 psiand 10 psi.

The fluidic device can include: a first pressure supply tube incommunication with a first pressurized gas source and connected to asealed headspace of the BGE reservoir through a first valve; a secondpressure supply tube in communication with the first pressurized gassource or a second pressurized gas source and connected, through asecond valve, to a sealed headspace of a sample reservoir in fluidcommunication with the sample channel; and a third pressure supply tubein fluid communication with one of the first pressurized gas source, thesecond pressurized gas source, and a pressure-reducing device, andconnected to a sealed headspace of a waste reservoir through a thirdvalve. The waste reservoir can be in fluid communication with a wastechannel (which may optionally be across from the sample channel), and influid communication with the separation channel. The method can furtherinclude: electronically opening and closing the first, second and thirdvalves in a defined sequence to flow the sample through the samplechannel, across the at least one SPE bed, and into the separationchannel.

The method can further include: capturing ions of the analyte componentin a mass spectrometer; detecting electronic signals corresponding tothe captured ions; and generating mass spectral informationcorresponding to the analyte component based on the electronic signals.

Still other embodiments are directed to microfluidic analysis systems.The systems include: a microfluidic device comprising at least oneseparation channel in fluid communication with a background electrolyte(BGE) reservoir, a sample channel in fluid communication with a samplereservoir and the separation channel and including at least one solidphase extraction (SPE) bed, and a waste reservoir in fluid communicationwith the separation channel. The systems also include: a first gassupply tube that connects a first pressurized gas supply to a sealedheadspace of the BGE reservoir through a first valve; a second gassupply tube that connects a second pressurized gas supply to a sealedheadspace of the sample reservoir through a second valve; and a thirdgas supply tube that connects a third pressurized gas supply or apressure-reducing device to a sealed headspace of the waste reservoirthrough a third valve. The waste reservoir is in fluid communicationwith a waste channel that fluidly connects the waste reservoir to theseparation channel. The systems also include: an electrode extendinginto the BGE reservoir so that when fluid is present in the BGEreservoir, the electrode contacts the fluid; and a controller inelectrical communication with a voltage source and with the first,second and third valves. The controller is configured so that duringoperation of the system, the controller directs the first, second andthird valves to open and close to flow a sample through the samplechannel, across the at least one SPE bed and into the separationchannel, and electrophoretically separate an analyte component in theseparation channel, without applying an electrokinetic voltage and/or avoltage gradient across the sample channel and/or SPE bed.

The sample channel can include at least one blocking member positionedadjacent to an end of the at least one SPE bed that is closest to theseparation channel to retain SPE material within the SPE bed.

The SPE bed has a length, measured along a flow direction defined by thesample channel, that can be between 100 μm and 1000 μm, and a volumethat can be between about 50 pL to about 10 nL.

The sample channel can be valveless so that the SPE bed is inuninterrupted fluid communication with the separation channel.

Some embodiments are directed to mass spectrometry systems that includea mass spectrometer and a microfluidic device onboard or incommunication with the mass spectrometer. The microfluidic deviceincludes at least one separation channel in fluid communication with abackground electrolyte (BGE) reservoir, a sample channel in fluidcommunication with the separation channel, a sample reservoir in fluidcommunication with the sample channel, a waste channel in fluidcommunication with the separation channel, a waste reservoir in fluidcommunication with the waste channel, and at least one electrosprayionization (ESI) emitter in fluid communication with the separationchannel. The sample channel includes at least one solid phase extraction(SPE) bed. The system further includes: a first conduit in communicationwith a first valve coupled to the BGE reservoir; an electrode extendinginto the BGE reservoir so that when fluid is present in the BGEreservoir, the electrode is in electrical communication with the fluid;a second conduit in communication with a second valve coupled to thesample reservoir; a third conduit in communication with a third valvecoupled to the waste reservoir; at least one voltage source electricallyconnected to the electrode; a first gas source in fluid communicationwith the first and second conduits; a second gas source or apressure-reducing device in fluid communication with the third conduit;and a controller in electrical communication with the first gas source,the second gas source or pressure-reducing device, and the at least onevoltage source. The controller is configured so that during operation ofthe system, the controller: (i) applies a pressure to a headspace of thewaste reservoir through the third conduit and concurrently applies apressure to a headspace of the sample reservoir through the secondconduit such that the pressure applied to the headspace of the wastereservoir is less than the pressure applied to the headspace of thesample reservoir; (ii) concurrently applies pressures to the headspacesof the sample reservoir and the BGE reservoir without applying a voltageto fluid in the sample reservoir or to fluid in the BGE reservoir; then(iii) reduces the pressure applied to the headspace of the samplereservoir so that the pressure then applied to the BGE reservoir isgreater than the pressure applied to the sample reservoir to transportthe sample and fluid from the BGE reservoir into the at least oneseparation channel; then (iv) applies an electric field along theseparation channel using the at least one voltage source toelectrophoretically separate an analyte component from the sample; andthen (v) perform electrospray ionization of the analyte component usingthe at least one ESI emitter to direct ions of the analyte componenttoward a collection device or an inlet of the mass spectrometer

The sample channel can include at least one blocking member positionedadjacent to an end portion of the at least one SPE bed that is closestto the separation channel.

The SPE bed has a length, measured along a flow direction of the samplechannel toward the separation channel, that can be between 100 μm and1000 μm, a volume that can be between about 50 pL and about 10 nL, andcan have a leading end positioned a distance of between 10 μm and 5 cmupstream from the separation channel.

The sample channel can be valveless so that the SPE bed is inuninterrupted fluid communication with the separation channel.

Still other embodiments are directed to fluidic devices that include: atleast one microfluidic or nanofluidic separation channel in fluidcommunication with a background electrolyte (BGE) reservoir; at leastone nanofluidic or microfluidic sample channel in fluid communicationwith a respective one of the at least one microfluidic or nanofluidicseparation channel; at least one solid phase extraction (SPE) bed in acorresponding one of the at least one nanofluidic or microfluidic samplechannels, positioned adjacent to a respective one of the at least onemicrofluidic or nanofluidic separation channels, so that a leading endof the at least one SPE bed is positioned between 10 μm and 5 cm fromthe separation channel; and at least one nanofluidic or microfluidicwaste channel that extends from a respective one of the at least oneseparation channel and is positioned across from a respective one of theat least one sample channel.

The device can include at least one blocking member positioned adjacentto an end of the at least one SPE bed that is closest to a respectiveone of the at least one separation channels so that it at leastpartially occludes a respective one of the at least one sample channels.

The leading end of the at least one SPE bed can be positioned at adistance of between 50 μm and 500 μm from the corresponding one of theat least one separation channels.

A width of the at least one SPE bed can be greater than a depth of theat least one SPE bed. The width being measured in a plane defined by thedevice and in a direction orthogonal to a flow direction defined by theat least one sample channel within the plane. The depth being measuredin a direction orthogonal to the plane and to the flow direction.

The at least one SPE bed has a length, measured along the flowdirection, that can be between 100 μm and 1000 μm, and a volume that canbe between about 50 pL and about 10 nL.

The device can include at least one electrospray ionization emitter influid communication with the at least one separation channel.

It is noted that aspects of the invention described with respect to oneembodiment, may be incorporated in a different embodiment although notspecifically described relative thereto. That is, all embodiments and/orfeatures of any embodiment can be combined in any way and/orcombination. Applicant reserves the right to change any originally filedclaim and/or file any new claim accordingly, including the right to beable to amend any originally filed claim to depend from and/orincorporate any feature of any other claim or claims although notoriginally claimed in that manner. These and other objects and/oraspects of the present invention are explained in detail in thespecification set forth below. Further features, advantages and detailsof the present invention will be appreciated by those of ordinary skillin the art from a reading of the figures and the detailed description ofthe preferred embodiments that follow, such description being merelyillustrative of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic illustration of a fluidic analysis systemaccording to embodiments of the present invention.

FIG. 1B is a schematic illustration of another embodiment of a fluidicanalysis system according to embodiments of the present invention.

FIGS. 2A-2H are schematic illustrations of a sequence of events for amicrofluidic device used to inject sample into a separation channelaccording to embodiments of the present invention.

FIGS. 3A-3F are schematic illustrations of the fluidic analysis systemshown in FIG. 1A, illustrating exemplary operative states of pressureand voltage inputs according to embodiments of the present invention.

FIGS. 4A-4F are schematic illustrations of fluidic devices according toembodiments of the present invention.

FIG. 5A is a schematic illustration of an example of a gas-tightconnection with a pressurized supply line for the background electrolyte(BGE) reservoir according to embodiments of the present invention.

FIG. 5B is a schematic illustration of another example of a gas-tightconnection with a pressurized supply line for the background electrolyte(BGE) reservoir according to embodiments of the present invention.

FIG. 6A is a schematic illustration of a fluidic device according toembodiments of the present invention.

FIGS. 6B and 6C are schematic, enlarged partial cutaway views of sampleflow channels with SPE bed blocking members according to embodiments ofthe present invention.

FIGS. 6D, 6E and 6F are schematic, enlarged cross-sectional views ofexemplary sample flow channels with blocking members according toembodiments of the present invention.

FIG. 7A is a schematic illustration of a microfluidic system accordingto embodiments of the present invention.

FIG. 7B is a schematic illustration of another embodiment of amicrofluidic system according to embodiments of the present invention.

FIG. 8 is an example of a timing chart for the pressures applied to thesample reservoir, the waste reservoir and the BGE reservoir forloading/injecting a sample into a separation channel according toembodiments of the present invention.

FIG. 9A is a schematic illustration of a portable MS device with anonboard microfluidic system that has pressure-driven injection accordingto embodiments of the present invention.

FIG. 9B is a schematic illustration of an external MS in communicationwith a microfluidic device according to embodiments of the presentinvention.

FIGS. 10A and 10B are flow charts of exemplary operations that can beused to carry out embodiments of the present invention.

FIG. 11 is a block diagram of a data processing system according toembodiments of the present invention.

FIGS. 12A-12C are electropherograms (BPI versus migration time inminutes) of a 4-peptide mix. FIG. 12A is an electropherogram of a CE-ESIof 5 μM sample. FIG. 12B is an electropherogram of an SPE-CE-ESI of 50nM sample. FIG. 12C is an electropherogram of an SPE-tITP-CE-ESI of 50nM sample. A and B correspond to unidentified trace components.

FIGS. 13A and 13B are electropherograms of a four peptide mix. FIG. 13Awas generated using electrokinetic (EK) injection and FIG. 13B wasgenerated using hydrodynamic injection according to embodiments of thepresent invention.

FIG. 14 is a graph of peak area ratio versus CE (capillaryelectrophoresis) migration time from the gated injection relative topeak areas for EK and hydrodynamic driven injections.

FIGS. 15A-15D are SPE-tITP-CE-ESI (for MS analysis) electropherograms ofincreasing concentrations of a four peptide mix according to embodimentsof the present invention.

FIGS. 16A-16C are plots of peak area, width and height versus sampleconcentration (nM) for SPE-tITP-CE-ESI analysis of Bradykinin accordingto embodiments of the present invention.

FIGS. 17A-17C are plots of peak area, width and height versus sampleconcentration (nM) for SPE-tITP-CE-ESI analysis of Thymopentin accordingto embodiments of the present invention.

FIGS. 18A-18C are plots of peak area, width and height versus sampleconcentration (nM) for SPE-tITP-CE-ESI analysis of peptide A accordingto embodiments of the present invention.

FIGS. 19A-19C are plots of peak area, width and height versus sampleconcentration (nM) for SPE-tITP-CE-ESI analysis of Angiotensin IIaccording to embodiments of the present invention.

FIGS. 20A-20C are plots of peak area, width and height versus sampleconcentration (nM) for SPE-tITP-CE-ESI analysis of Met-Enkephalinaccording to embodiments of the present invention.

FIGS. 21A-21C are plots of peak area, width and height versus sampleconcentration (nM) for SPE-tITP-CE-ESI analysis of Peptide B accordingto embodiments of the present invention.

FIG. 22 are electropherograms (BPI versus migration time (minutes)) ofphosphorylase B tryptic digests separated using CE-ESI (top and enlargedinset) and SPE-tITP-CE-ESI (bottom).

FIG. 23 is an electropherogram of SPE-tITP-CE-ESI separation of 0.5mg/mL E. coli digest (BPI versus migration time, minutes) according toembodiments of the present invention.

FIGS. 24A and 24B are electropherograms of SPE-tITP-CE-ESI MSillustrating desalting capability of the integrated SPE-tITP-CE-ESImicrofluidic devices contemplated by embodiments of the presentinvention.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention will now be described more fully hereinafter withreference to the accompanying figures, in which embodiments of theinvention are shown. This invention may, however, be embodied in manydifferent forms and should not be construed as limited to theembodiments set forth herein. Like numbers refer to like elementsthroughout. In the figures, certain layers, components or features maybe exaggerated for clarity, and broken lines illustrate optionalfeatures or operations unless specified otherwise. In addition, thesequence of operations (or steps) is not limited to the order presentedin the figures and/or claims unless specifically indicated otherwise. Inthe drawings, the thickness of lines, layers, features, componentsand/or regions may be exaggerated for clarity and broken linesillustrate optional features or operations, unless specified otherwise.The abbreviations “FIG. and “FIG.” for the word “Figure” can be usedinterchangeably in the text and figures.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms, “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises,”“comprising,” “includes,” and/or “including” when used in thisspecification, specify the presence of stated features, regions, steps,operations, elements, and/or components, but do not preclude thepresence or addition of one or more other features, regions, steps,operations, elements, components, and/or groups thereof. As used herein,the term “and/or” includes any and all combinations of one or more ofthe associated listed items. As used herein, phrases such as “between Xand Y” and “between about X and Y” should be interpreted to include Xand Y. As used herein, phrases such as “between about X and Y” mean“between about X and about Y.” As used herein, phrases such as “fromabout X to Y” mean “from about X to about Y.”

It will be understood that when a feature, such as a layer, region orsubstrate, is referred to as being “on” another feature or element, itcan be directly on the other feature or element or intervening featuresand/or elements may also be present. In contrast, when an element isreferred to as being “directly on” another feature or element, there areno intervening elements present. It will also be understood that, when afeature or element is referred to as being “connected”, “attached” or“coupled” to another feature or element, it can be directly connected,attached or coupled to the other element or intervening elements may bepresent. In contrast, when a feature or element is referred to as being“directly connected”, “directly attached” or “directly coupled” toanother element, there are no intervening elements present. Althoughdescribed or shown with respect to one embodiment, the features sodescribed or shown can apply to other embodiments.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the present applicationand relevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. Well-known functions orconstructions may not be described in detail for brevity and/or clarity.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if the device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of over and under. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

It will be understood that, although the terms first, second, etc. maybe used herein to describe various elements, components, regions, layersand/or sections, these elements, components, regions, layers and/orsections should not be limited by these terms. These terms are only usedto distinguish one element, component, region, layer or section fromanother region, layer or section. Thus, a first element, component,region, layer or section discussed below could be termed a secondelement, component, region, layer or section without departing from theteachings of the present invention.

The term “about” means that the stated number can vary from that valueby +/−20%.

The term “analyte” refers to a molecule or substance undergoinganalysis, typically, at least for mass spectrometry analysis, having anion or ions of interest in a mass-to-charge (m/z) range of interest. Theanalyte can comprise biomolecules such as polymers, peptides, proteinsand the like. Embodiments of the invention are particularly suitable foranalyzing intact monoclonal antibodies. Embodiments of the invention areparticularly suitable for analyzing metabolites.

The term “pre-concentration” refers to analytes at increasedconcentration relative to a concentration at introduction to a fluidicanalysis device or system so that a sample with the analytes isprocessed, typically prior to introduction into a separation channel, tocontain analytes at higher concentrations relative to concentration(s)when introduced to the system, device, or process, i.e., as introducedto a sample channel or reservoir upstream of the SPE bed and/orseparation channel. The term “pre-concentrating” refers to processes,typically on-chip processes, that achieve the pre-concentration. Theterm “focusing” refers to performing the pre-concentrating usingelectrokinetic techniques.

The term “microchip” refers to a substantially planar, thin, and, insome embodiments, rigid fluidic device. The term “thin” refers to athickness dimension that is less than about 10 mm, typically about 1 mmor less. The microchip typically has a width and length that is lessthan about 6 inches and a thickness that is less than about 5 mm,typically between about 2000 μm to about 250 μm.

The terms “integrated” and “integral” and derivatives thereof means thatthe component and/or process or action is incorporated into or carriedout by a fluidic device. For example, an integrated SPE-tITP-CE-ESImicrofluidic device has an onboard SPE bed, a CE channel, at least onean ESI emitter can perform tITP.

The term “in-line coupling” refers to the inclusion of a solid phaseextraction (SPE) sorbent(s) proximate or adjacent an entry to the CEcapillary (also called a separation channel), typically via a side orcross-channel at a head or ingress end portion of the CE capillary.

The term “SPE bed” refers to a segment of a fluid channel that holds avolume of SPE material (i.e., sorbent) in a suitable density so thatsample flows through the bed prior to entering the separation channel.

The term “high voltage” refers to voltage in the kV range, typicallybetween about 1-100 kV, more typically between about 1-20 kV. ESIprocesses can employ potentials of a few kVs, typically between about 1kV to about 5 kV, for example. Other voltages may be appropriate.

The term “microfluidic” refers to fluid flow channels that havesub-millimeter or smaller size width and/or depth (e.g., the termincludes nanometer size channels) and includes channels with widthand/or depth dimensions in a size range of about tens to hundreds ofmicrons.

The term “hydrodynamic driven injections” refers to pressurecontrollably applied to one or more fluid channels to transport a targetsample for analysis to a separation channel without requiring voltage.

All of the document references (patents, patent applications andarticles) are hereby incorporated by reference as if recited in fullherein.

In typical free zone capillary electrophoresis (CE) experiments, asample plug is injected into a column, and an applied electric fieldcauses sample components to separate according to differences in theirmobilities. The mobility of a molecule is the sum of its electrophoreticmobility and the electroosmotic mobility, and any driven flow, ifpresent, of the separation column.

The term “plug” with respect to “sample” refers to a quantity of asample collected/localized within a spatial region, such as within aspatial region of a carrier fluid. The plug can be a physical band orsegment with defined leading and trailing ends so that there is adistinct clearance between successive plugs or bands.

The term “separated sample” refers to the electrophoretically separatedsample and/or components of a sample mixture (i.e., spatially separatedalong the axial extent of the separation channel) and may or not beseparated into individual components. Components will be separated basedupon their effective electrophoretic mobilities and separation ofcomponents will depend on the difference in effective mobilities. Theseparated sample can be detected by observing the spatial separation inthe separation channel and/or by observing their arrival times at theelectrospray emitter or detector. Effective electrophoretic mobility isdefined as the observed velocity in the separation channel divided bythe electric field strength in the separation channel and will includethe actual electrophoretic mobility and the vector sum of any othereffect imparting velocity to the component including, but not limitedto, electroosmotic or pressure driven transport. The “sample” cancomprise a collection of one or more different components (i.e., ananalyte and surrounding matrix material). The sample is introduced intothe fluidic device. During separation, an “analyte component(s)” of thesample can be separated for analysis from other components.

The analyte in a sample can be any analyte of interest including, forexample, various mixtures including synthetic and biologicalmacromolecules, nanoparticles, small molecules, DNA, nucleicacids/polynucleic acids, peptides, proteins and the like. The sample caninclude one or more polar metabolites such as amino acids or chargedmolecules, molecules, peptides, and proteins. The sample may also oralternatively include molecules extracted from biofluids, blood, serum,urine, dried blood, cell growth media, lysed cells, beverages or food;or environmental samples such as water or soil.

Low pH refers to acidic levels (below 7) and high pH refers to basiclevels (above 7).

The term “low organic” refers to levels at or below 25% by volume and“high organic” refers to levels above 25% by volume.

With respect to certain processing conditions associated with SPE, lowsalt content refers to between 0-0.1 M salt content, while high saltcontent can refer to above 0.1, such as above 0.1 M to about 1 M saltcontent.

Referring to FIG. 1A, an exemplary analysis system 100 with a fluidicdevice 10 at a controller 100 c is shown. The controller 100 c caninclude at least one processor that can be configured to direct thesystem 100 to operate with a sequence of operations, such as a sequenceof voltage and/or pressure inputs to components of the fluidic device10. The fluidic device 10 can be a microchip 10 c. The fluidic device 10can include at least one separation channel 25 (which can also beinterchangeably referred to as a capillary channel) and at least onesolid phase extraction (SPE) bed 28 in fluid communication with theseparation channel 25.

The SPE bed 28 can reside in a side channel 31 that connects to anupstream, upper end portion or ingress end portion 25 u of theseparation channel 25. The side channel 31 can be orthogonal to theseparation channel 35 or may reside at other angles (see, e.g., FIG.4D). The SPE bed 28 can reside between the separation channel 25 and areservoir 30. The reservoir 30 can be configured to supply definedfluids, such as a sample fluid for analysis, to the SPE bed 28, whichflows over or through the SPE bed 28, then to the separation channel 25.The SPE bed 28 can be pre-packed or formed prior to an analysis, such asprovided in a device ready for use, or may be packed in situ prior toand/or as part of an analysis. The fluidic channel 31 holding at leastone SPE bed 28 can include at least one blocking member 128, such as aweir 128, or membrane. In some embodiments, the blocking member 128 canbe other fluid permeable, which may be particularly suitable forembodiments where the blocking member 128 extends totally across thesample inlet channel 31. The blocking member 128 is configured to allowfluid, such as a liquid or gas sample S to flow through or over the SPEbed 28 while inhibiting SPE material from the bed 28 from entering theseparation channel 25. The term “weir” refers to structures with anobstruction across a width or partial width of the sample inlet channel31. In some embodiments, the blocking member 128 resides between the endof the SPE bed 28 e and the separation channel 25, typically adjacentthe separation channel, such as, for example, within about 10 μm tounder 5 cm, typically between 10 μm to about 500 μm, and more typicallybetween about 50 μm to about 500 μm from the separation channel 25.

As shown in FIG. 6A, the side channel 31 (which can also be referred tointerchangeably as “a sample inlet channel”) can be configured to haveseparate segments that merge into the sample inlet channel with the atleast one blocking member 128. If so, separate reservoirs 30 ₁, 30 ₂ mayconnect to a different branch 31 a, 31 b. The branches 31 a, 31 b canfluidly merge into the channel 31. One reservoir 30 ₁ can comprise thesample S and another 30 ₂ can hold SPE material 28 s and/or other fluidsfor processing (e.g., wash or solvent and the like). Eachbranch/reservoir pair 30 ₁/31 b, 30 ₂/31 a can serially flowablyintroduce material to the inlet side channel 31.

The blocking member 128 can extend down from the ceiling 31 c (128 ₁,FIG. 6B) or up from the floor 31 f (FIGS. 2B, 6F) and/or extend inwardlyfrom sidewalls 31 s (FIGS. 6D, 6E). The at least one blocking member 128can be a plurality of blocking members 128 ₁, 128 ₂ (up to 128 n, wheren=1 to 10) extending from two of the ceiling 31 c, floor 31 f orsidewalls 31 s (128 ₁, 128 ₂, FIG. 6B, 128 ₁-128 ₄, FIGS. 6E, 6D). Theat least one blocking member 128 can have a height that is less than adepth dimension of the channel 31, typically between about 30-70% of thedepth dimension. For example, for a channel 31 with a depth dimension of100 μm, the height of the blocking member 128 can be between 30 and 70μm, such as about 30 μm, about 40 μm, about 50 μm, about 60 μm, andabout 70 μm. As shown in FIG. 6B, where two or more blocking members 128₁, 128 ₂ are used for a respective bed 28, the two can be offset yetclosely spaced apart (typically between about 1-20 μm) from each other.In other embodiments, at least two of the blocking members 128 ₁, 128 ₂can be directly aligned from each other as shown in FIG. 6C.

FIGS. 6D-6F illustrate examples of sample channel geometries which maybe formed by etching, milling, molding and the like. The number andplacement of the at least one blocking member 128 are by way ofillustration only.

As shown in FIG. 6A, the fluidic device 10 comprises a planar rigid,semi-rigid or flexible (polymeric) ceiling 10 c that is sealablyattached to a substrate 10 s holding the fluid channels 21, 25, 31, 32,typically in multiple sets, to seal the fluid channels. The blockingmember(s) 128 can be integrally formed in the sample inlet channel 31 orbe attached as a separate component, where more than one are usedcombinations of integral and separate components can be used.

The SPE bed 28 can comprise any suitable solid phase extraction materialand/or sorbent including mixtures of different materials. The SPE bed 28can comprise, for example, small particles having a diameter and/or amaximal height/width or length dimension that is less, typically atleast 30% less, than the width/depth of the fluidic channel 31 holdingthe bed 28. The SPE bed 28 can comprise 5 μm diameter porous Oasis HLBparticles. Other particle types suitable for SPE beds 28 include, butare not limited to, reverse phase, ion exchange, immuno sorbent, HILIC,normal phase and mixed mode. An example of mixed mode is Water's OasisHLB particles. A few examples of each type (to be clear this is not anexhaustive list) are provided below.

-   Revered Phase—C4, C8, C18, Phenyl-   HILIC—bare silica-   Ion Exchange—Titanium Dioxide for phosphopeptides,-   Affinity—antibody, lectins for glycopeptides, Nickle-His tag

In some embodiments, the SPE material for the SPE bed 28 can include aretentive reversed-phase material and/or a mixed mode stationary phase,such as a combined reversed-phase retention with ion exchange retention.See, e.g., Gilar et al., Journal of Chromatography A 2008, 1191,162-170; and Li et al., Talanta 2015, 140, 45-51. The contents of thesedocuments are hereby incorporated by reference as if recited in fullherein.

The SPE bed 28 can have any suitable size, density and volume. In someembodiments, the length of the SPE bed 28 can be between 1 μm and 1 cm,typically between 100 μm and 1000 μm, such as about 100 μm, about 200μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700μm, about 800 μm, about 900 μm and about 1000 μm long. The length of theSPE bed 28 can be less than the fluidic channel 31 in which it resides,typically between 2-1000× less. The SPE bed 28 typically resides closerto separation channel 25 than the reservoir 30. The leading end (the endclosest the separation channel 25) of the SPE bed 28 can reside withinabout 10 μm to under 5 cm, typically between 10 μm to about 500 μm, andmore typically between about 50 μm to about 500 μm from the entry orboundary of the separation channel 25. The width of the fluid channel 31holding the SPE bed 28 can be greater than the depth, typically 2-200×greater. The volume of a (packed) SPE bed 28 can be between about 50 pLto about 10 nL, such as about 425 pL, in some embodiments.

The SPE bed 28 can be packed in the sample channel 31 prior to analysisof a sample S. An SPE slurry can be flowably introduced into the samplechannel 31. The term “slurry” refers to a viscous solution of SPEmaterial. The viscosity of the SPE slurry is greater than the viscosityof other liquids used for the analysis.

In some embodiments, a low concentration of an ion-pairing agent such asTrifluoroacetic acid (TFA) can be introduced into the fluid channel 31and the SPE bed 28 to retain a sample S. Where TFA is used, it can beprovided in a concentration between about 0.01% to about 1% by volume.This agent can be introduced before and/or with the introduction of asample into the channel 31. The TFA can be removed (e.g., washed intothe waste channel 32 and/or waste reservoir 35) prior to elution forintroducing the sample as a sample plug in the separation channel. AnyIon-Pairing agent current used in chromatography may be used for SPEwith this device, depending on the chemistry of the SPE sorbent and theidentity of the sample. Ion-exchange chromatography systems havepreviously been utilized in HPLC analysis of ionic samples includingphase partition chromatography using ion-pair reagents. The ionicsamples form an ion-pair with ion-pair reagents in the mobile phase tobecome electrically neutral. The increase in hydrophobic character ofthe ion-pair results in a greater affinity for the reverse stationaryphase and leads to sample resolution.

See, e.g., the below webpage with different examples of ion-pairingagents:http://www.teichemicals.com/eshop/en/us/category_index/00418/#innerlink_10_1_1,the contents of which are hereby incorporated by reference as if recitedin full herein.

The ion-pairing agent can be pre-loaded into the fluid channel 31 (oronto the material of the SPE bed 28) or added in situ during or justprior to an analysis, for example.

Still referring to FIG. 1A, the system 100 can include a voltage supply95, which may be a high voltage supply. As shown, the system 100 canalso include at least one pressurized gas source 90 in communicationwith the BGE reservoir 20 and the side reservoir 30 via supplylines/conduits 70 and respective valves 120, 130. The BGE reservoir 20can feed a BGE channel 21 that is connected to the separation channel25.

As shown, the system 100 can include a pressure reducing device 91 incommunication with a waste reservoir 35 which may also be connected viaa respective valve 135. The pressure-reducing device 91 can have anactive or passive configuration, i.e., can comprise a vacuum, a pump, anevacuated reservoir, or any other enclosed volume at a pressure lessthan the pressure applied to the BGE reservoir 20 and/or the sidereservoir 30, typically less than ambient pressure, that will reduce thepressure in the headspace of the waste reservoir 35 once connected.

While shown as a separate device 91, the pressure reducing device 91 canbe configured with a supply line 70 to connect the waste reservoir 35 tothe first pressurized gas source 90 and pressure can be controlled toprovide the desired input. Also, the pressure reducing device 91 is notrequired to be a vacuum and may operate at different pressures toprovide the desired operational sequence as will be discussed furtherbelow.

The waste reservoir 35 can be in fluid communication with the separationchannel 25. The waste reservoir 35 can reside in a second side channel32. The waste channel 32 can reside across from the side channel 31 withthe SPE bed 28. The waste channel 32 can be laterally in line with theside channel 31 (i.e., FIG. 4A) or can be longitudinally offset from theside channel 31 (i.e., FIG. 4C).

The background electrolyte (BGE) reservoir 20 can reside at a top abovethe separation channel 25. The BGE reservoir 20 can reside directlyadjacent the separation channel or may have a BGE flow channel 21 thatmerges into the separation channel 25 to position the BGE reservoir 20 adistance away from the sample channel 31 and the sample waste channel32.

In the embodiment shown in FIG. 1A, the fluidic device 10 has crosschannels defined by the sample channel 31 and the sample waste channel32, which can reside on opposing sides of the separation channel 25 andmay optionally be orthogonal to and extend across to intersect theseparation channel 25.

In some embodiments, defined pressures can be supplied by thepressurized gas supply 90 and/or 91 to the respective gas supply lines70 which are sealably attached to respective reservoirs 20, 30, 35,typically conduits or lengths of tubing 70 from at least one pressurizedgas source 90. The pressurized gas of the gas supply 90 for providingpressure-driven injection can comprise air and/or noble gases such ashelium or nitrogen or other inert gases. FIG. 1A illustrates discretevalves 120, 130, 135 for the gas supply lines 70 ₂, 70 ₁, 70 ₃respectively. Any or all of valves 120, 130, 135 can be three-wayvalves.

It is also noted that while the system 100 is shown with threereservoirs 20, 30, 35, less or more reservoirs and, indeed, sidechannels, may be used.

FIG. 1A also illustrates that the fluidic device 10 can include a pump40 and at least one electrospray ionization (ESI) emitter 50 that canspray a separated sample 50 s for analysis. The pump 40 can include achannel 40 c that extends to a junction 40 j adjacent the ESI emitter50. The electrospray 50 s from the at least one emitter 50 can beprovided to a collection device 202 (FIG. 7A) for subsequent analysisand/or toward an entrance aperture/inlet of a mass spectrometer 200 witha detector (FIG. 1A). The pump can optionally comprise an electroosmotic(EO) pump.

FIG. 1B illustrates that the analysis system 100 can include the fluidicdevice 10 with the at least one SPE bed 28 and may also include at leastone detector 1200 to obtain signal from the sample in the separationchannel 25. The fluidic device 10 may be configured without the at leastone ESI emitter 50 and may be used without requiring the input to themass spectrometer 200. The detector 1200 can be an electronic detectorsuch as an optical detector and/or a conductance detector (i.e.,comprising an ammeter), for example. Where used, the optical detector1200 can comprise an avalanche photodiode and laser, or photomultiplierand convention light source such as a blackbody source or dischargesource, or any combination of the above as is well known to those ofskill in the art. The detector 1200 can obtain signal for qualitativeand/or quantitative data of a sample in the separation channel 25. Insome embodiments, the analysis system 100 can include both the at leastone detector 1200 and the at least one ESI emitter 50 for input to theinlet/entrance aperture of the mass spectrometer 200. In someembodiments, both mass spectrometer detection and optical detection bythe detector 1200 can be carried out simultaneously, i.e., signal fromthe ESI emitter 50 the inlet of the mass spectrometer 200 can beobtained while signal from the detector 1200 is obtained for arespective sample, for example.

The separation channel 25 is shown in FIGS. 1A and 1B as having aserpentine shape but other configurations may be used. For example, thegeometry of the separation channel 25 can be straight or curved, and thecross-sectional profiles of the channels do not all have to be the same.For further discussion of exemplary microfluidic devices, see, e.g.,U.S. patent application Ser. Nos. 14/001,549 and 14/368,971, thecontents of which are hereby incorporated by reference herein.

The fluidic (sample) channel 31 holding the SPE bed 28 can have a widthand/or depth that is between 40 nm and 1000 μm, more typically betweenabout 1 μm and about 100 μm, such as a channel depth and width of about10 μm (depth) and 70 μm (width), respectively. As used herein, the“width” of channel 31 is measured in the plane of device 10 (i.e., inthe plane defined by the microfluidic chip) and in a direction that isperpendicular to an axis of channel 31, where fluid flow occurs throughchannel 31 in a direction parallel to the axis. As used herein, the“depth” of channel 31 is measured in a direction perpendicular to theplane of device 10 and to the direction along which the width ismeasured. The fluidic channels 21, 31 and 32 can all have the same depthor may have different depths. The fluidic channels 21, 31 and 32 canhave the same width or different widths. The separation channel 25 canhave any suitable length, typically between 1 cm to 100 cm, moretypically between about 20-30 cm, such as about 23 cm.

As shown in FIGS. 1A and 1B, for example, the sample channel 31 isvalveless so that the SPE bed 28 remains in fluid communication with theseparation channel 25 continuously, i.e., the fluid device 10 does notrequire any valve to isolate the SPE bed 28 from the separation channel25. Stated differently, the at least one SPE bed 28 is held in thesample channel 31 and in the sample flow path without requiring anyvalve to isolate the SPE bed 28 during in-line operation.

The BGE reservoir 20 can be at the top of the separation channel 25(directly or via the BGE channel 21). The BGE channel 21 can have alength “L₁” (FIGS. 1A, 1B) extending from the BGE reservoir 20 to theSPE bed channel 31 (which can also be called the “sample channel” as thesample is introduced into this channel to contact the SPE bed 28 priorto entering the separation channel 25) and/or the channel 31 and wastechannel 32 cross/intersection with the separation channel 25. The length“L₁” can be any suitable length, such as, for example, between 1-200 mmlong. The SPE bed channel 31 can have a length “L₂” (FIGS. 1A, 1B) andthe waste channel 32 can have a length “L₃” (FIGS. 1A, 1B). Also, thelengths L₁, L₂, L₃ of one or more of the channels 21, 31, 32, can be anysuitable length such as about 1 mm, about 5 mm, about 10 mm, about 20mm, about 30 mm, about 40 mm, about 50 mm, about 60 mm, about 70 mm,about 80 mm, about 90 mm, or about 100 mm, in some embodiments, butother lengths can be used. In general, the “length” of a channel (suchas the lengths L₁, L₂, and L₃ referenced above) is measured along anaxis of the channel in a plane of device 10 (i.e., in the plane definedby the microfluidic chip), where fluid flow occurs through the channelin a direction parallel to the axis. Where used, the injection crossconfiguration may be such that channels 21, 31 and 32 have substantiallythe same length or different lengths, but typically lengths that aremuch less than the length of the separation channel 25. The sample andsample waste channels 31, 32, can be longer or shorter than the BGEchannel 21 and may, for example, be between 1 mm and 5 cm, typicallybetween about 1-100 mm long. In some embodiments, the sample and samplewaste channels 31, 32 are between about 1-20 mm, such as about 8 mm inlength.

One or both of the reservoirs 20, 30 can be in fluid communication withone or more external fluid sources to provide fluid thereto duringanalysis and/or one or both of the reservoirs 20, 30 may be pre-loadedprior to active analysis.

Still referring to FIG. 1A, in some embodiments, a fluid junction 40 jcan be used to connect the separation/transfer channel 25 and respectivepump channel 40 c. The fluid junctions can be nanojunctions with theassociated nanojunction channels having nanometer-sized depths. The pumpchannel 40 c and/or separation channel 25 can have micrometer-sizedwidths. The junctions 40 j can have, for example, a depth of about 50 nmand a width of about 50 μm. The depth of the channel may be dictated bythe ionic strength of the buffers used in the experiment/analysis andthe corresponding Debye lengths. Nanochannel depth should be on theorder of the Debye length or smaller.

FIGS. 2A-2H and 3A-3F, illustrate an exemplary sequence of operationsthat can be carried out according to some embodiments of the presentinvention. FIGS. 2A and 2B show that a fluidic device 10 can be providedand a SPE bed 28 can be loaded or pre-loaded for use. FIG. 2B shows thefluidic channel 31 with the SPE bed 28.

FIGS. 2C and 3A illustrate a pre-conditioning of the SPE bed 28 can becarried out. FIG. 2C shows a pre-conditioning fluid P such as a solventfrom the reservoir 30. FIGS. 2C and 3A illustrate a pressure drivinginput P1 applied to the reservoir 30 while a pressure reducing deviceapplies a reduced pressure such as a vacuum V1 to the waste reservoir 35and voltage is OFF. FIG. 3A illustrates the valves 130 and 135 are ONwhile valve 120 is OFF and the voltage is OFF (i.e., not applied toinputs 20 or 40). While the pressure is shown as OFF for valve120/pressure input to reservoir 20, a pressure may be applied but notsufficient to interfere with the operational inputs. Thepre-conditioning fluid P from the reservoir 30 flows across/through theSPE bed 28 and into the waste channel 32, then to the waste reservoir35. BGE solution B can flow from the BGE reservoir 20. The flowdirection of the pre-conditioning fluid P and BGE fluid B are indicatedby the flow direction arrows in FIG. 2C.

The term “pre-condition” refers to exposing the SPE bed 28 to anysolvent or liquid mixture of an appropriate organic content, pH, and/orsalt content. The pre-conditioning agent or mixture may change based onthe chemistry of the SPE sorbent and the identity of the sample S. Thepre-conditioning material can be a defined liquid that can: optionallya) wet the SPE bed (although this is less of a concern with themicrochip if the bed has been wetted during the chip filling step)and/or b) condition the SPE bed 28 to be in a desired current solventcondition that is suitable/appropriate for binding the target sample S.For example, in reversed phase SPE, loading can occur under low organicconditions. Therefore, the pre-conditioning step can be used to ensurethat the SPE bed 28 is wetted or exposed to low organic solvent. Asanother example, loading using a HILIC stationary phase occurs underhigh organic conditions. Loading with ion-exchange may occur under lowsalt, while elution occurs at high salt. In some embodiments, pH canalso affect loading and elution.

FIGS. 2D and 3B illustrate a sample loading step where sample S isintroduced via reservoir 30 into the fluidic channel 31 with the SPE bed28. While the device 10 shows the sample S introduced via the samereservoir 30 as the pre-conditioning material, a separate reservoir canbe used (not shown). The sample loading can be carried out using onlypressure inputs such as an applied pressure P1 and a reduced pressuresuch as a vacuum V1, with voltage OFF. FIG. 3I illustrates the valves130 and 135 are ON while valve 120 is OFF (closed to supply line 70 intoreservoir 20) and the voltage is OFF (i.e., not applied to inputs 20 or40). FIGS. 2E and 3C illustrate an optional wash step where pressure P1is applied to the reservoir 30 (or another reservoir in fluidcommunication with channel 31) and a lower pressure, typically a vacuumV1, is applied to the waste reservoir 35, with voltage OFF, to introducea wash solvent to the fluidic channel 31 across the SPE bed 28 and intothe waste reservoir 35. FIG. 3C illustrates the valves 130 and 135 areON while valve 120 is OFF and the voltage is OFF (i.e., not applied toinputs 20 or 40).

The wash fluid, like the pre-conditioning material, can vary based onthe chemistry of the SPE sorbent and the identity of the analyte(s). Thepurpose of the wash fluid is to displace the salt and any unboundspecies without unduly affecting the retention of the analyte(s) ofinterest. As an example, in reversed phase SPE, loading and bindingoccurs under low organic content conditions. Therefore, a low organicsolution can be used to wash the sample S. In many cases, the washsolvent and the loading solvent can be the same. As with thepre-conditioning material/solvent, the washing fluid can change based ondifferent mechanisms, HILIC, ion-exchange, and the like as is well knownto those of skill in the art.

FIGS. 2F and 3D illustrate an elution operation that can be carried outafter the washing and/or sample loading step to distribute a sample plugSp into the separation channel 25. Pressure P1 is applied to thereservoir 30 with an elution fluid E while pressure P2 is also appliedto the BGE reservoir 20 and no reduced pressure such as a vacuum isrequired to be applied to the waste reservoir 35 (i.e., no vacuum isapplied but a positive pressure may be applied to help keep the sampleplug Sp in the separation channel 25). FIG. 3D illustrates the valves120 and 130 are ON while valve 135 is OFF and the voltage is OFF (i.e.,not applied to inputs 20 or 40). The elution fluid E forces the sampleplug Sp into the separation channel 25 and may flow into the wastechannel 32 as indicated by FIG. 2F.

The elution material E can comprise high or low organic content. As usedabove, the term “low organic content” refers to an elution material inwhich organic analyte substances collectively comprise 25% or less ofthe volume of the elution material, and “high organic content” refers toan elution material in which organic analyte substances collectivelycomprise more than 25% by volume of the elution material.

The elution material E can comprise high or low salt content. Asdiscussed above, with respect to certain processing conditionsassociated with SPE, low salt content refers to an elution material inwhich a concentration of dissolved salts is between 0-0.1 M, while highsalt content refers to an elution material in which a concentration ofdissolved salts is above 0.1 M, such as above 0.1 M to about 1 M.

The elution material E can have high or low pH. As used herein, low pHrefers to acidic pH levels (below 7), and high pH refers to basic pHlevels (above 7).

The elution material E can comprise a combination of salts, and organicanalyte substances, and the organic content and salt content can each behigh or low, independent of the other. Further, the elution material canhave a high or low pH, independent of the salt and organic content.

In affinity chromatography/affinity SPE, the elution material may be anagent that disrupts the affinity binding, such as an analog of thesample with a higher binding affinity for the sorbent material. Theelution and/or tITP material can be introduced via the BGE reservoir andchannel 20/21 and/or via the sample channel 31 and a reservoir in fluidcommunication therewith.

FIGS. 2G and 3E illustrate an optional clearing operation that can beused according to embodiments of the present invention. Pressure inputP1 can be reduced or removed while pressure input P2 is applied to theBGE reservoir 20, with voltage OFF. Pressure input to the wastereservoir 35 can also be reduced or removed so that BGE fluid flows downto push the sample plug Sp past the fluidic channel 31 with the SPE bed28. The BGE fluid can push the elution fluid E to exit the SPE bed 28toward the reservoir 30. FIG. 3E illustrates the valve 120 is ON whilevalves 130 and 135 are OFF and the voltage is OFF (i.e., not applied toinputs 20 or 40).

FIGS. 2H and 3F illustrate that a subsequent transport/separationbetween successive sample plugs Sp can be carried out by applyingvoltage to the BGE reservoir 20 and to the ESI orifice without applyingpressure to reservoir 30 or 35. The applied voltage can be high voltageat reservoir 20 and a positive voltage at the ESI orifice (e.g., at EOpump input 40) to drive the sample plug Sp to the ESI emitter orifice50. FIG. 3F illustrates the valves 120, 130 and 135 are OFF and thevoltage is ON (i.e., applied to inputs 20 or 40). As discussed above,the term “high voltage” refers to voltage in the kV range, typicallybetween about 1-100 kV, more typically between about 1-20 kV. ESIprocesses can employ potentials of a few kVs, typically between about 1kV to about 5 kV, for example. Other voltages may be appropriate.

Generally stated, in some embodiments of the present invention, pressure(alone) can be used to inject samples of a microfluidic device 10 formicrochip capillary electrophoresis (CE). The pressure-drive method hasadvantages over other microfluidic injection methods such asvoltage-driven loading methods, in that it can use a simple channelgeometry, but is capable of generating desired sample plug Sp sizes(FIGS. 2F, 2G, 2H) by simply adjusting the time the pressure is appliedand/or the amount of pressure applied to the reservoirs 20, 30. Thepressure-drive operation is also typically free of eletrokineticinjection bias and no voltage is required to be applied to the samplereservoir 30.

In some embodiments, the pressure applied concurrently to the BGEreservoir 20 and the sample reservoir 30 for the injection (FIGS. 2F,3D) is between 1 and 30 psi for between 1-5 seconds, more typicallybetween 1 and 12 psi. Then, for the clearing of the tail end of thesample (FIGS. 2G, 3E), the pressure P2 in the BGE reservoir 20 can beheld the same or reduced by 10-80% and the pressure in the reservoir 30can be reduced more than the reduction in the pressure of the BGEreservoir 20, e.g., typically so that it is less than 0.1 psi, e.g.,zero or at ambient or atmospheric pressure or below ambient oratmospheric pressure (e.g., under vacuum). The term “reduced” withrespect to pressure can also include removing the applied pressurealtogether.

The clearing pressure on the BGE reservoir 20 (alone) can be held for atime that is less than the injection time where pressure is applied toboth reservoirs 20, 30 (FIGS. 2F, 3D). The clearing time for thepressure applied only to the BGE reservoir 20 can be 2 seconds or less,1 second or less or 0.5 seconds, for example.

However, while pressure-driven loading and injection may be particularlyuseful, embodiments of the invention can include electrokinetic (EK)gate methods using a sequence of different voltages applied to themicrofluidic device 10 for sample loading and/or injection.

In some embodiments, pressure-driven methods can be particularlysuitable for performing online sample concentration methods such astransient isotachophoresis (tITP), because sample plugs Sp withsignificantly different properties (electrical conductivity, pH, orviscosity) compared to the background electrolyte (BGE) can be injected.Salt or other electrolyte material in the sample/sample reservoir 30 canbe used for tITP. The pressure-driven operation can position awell-defined band of sample (sample plug Sp) into the separation channel25 of the microfluidic device using only pressure-driven flow and canalso be used for online sample focusing methods that are not possible byother microfluidic injection methods.

Referring to FIGS. 1 and 7A, head pressure can be applied to twodifferent fluid reservoirs 20, 30 and/or 35 on or in communication withthe microfluidic device 10, typically using off-device (e.g., off-chip)on/off valves 120, 130, 135. The term “head pressure” refers to the gaspressure in a sealed headspace of the reservoir above the liquid. Thehead pressure of the BGE reservoir 20 is labeled P2 and the headpressure of the sample reservoir 30 is labeled N. A controller 100 c canbe in communication with the valves 120, 130, 135 to independentlycontrol when the pressures P1, P2 and V1 are applied at the respectivereservoirs 20, 30, 35. Thus, for sample loading no voltage is requiredto be applied to either the BGE reservoir 20 or the sample reservoir 30(FIGS. 2D, 2F, 2G, for example).

The microfluidic channels 25, 31, 32 within the device 10 can, in someembodiments, be configured to form a simple injection cross.

In some embodiments, the sample waste channel 32 may be excluded asshown in FIG. 4B, for example. Thus, the use of a “tee” intersection ofthe sample channel 31 (in lieu of the cross channel configuration) tothe separation channel 25 may be used and may be implemented using arelatively precise pressure on the BGE reservoir 20 to hold that fluidstationary for injection/sample loading, at least where EK drive is notemployed.

Referring to FIGS. 2F, 3D, pressure is concurrently applied to thesample reservoir 30 (P1) and the BGE reservoir 20 (P2) to drive sample Sfrom the sample reservoir 30 (shown by the directional arrows) into theseparation channel 25 from the sample channel 31, and typically thesample waste channel 32, but not the BGE channel 21. When a plug of asample Sp in the separation channel 25 reaches a desired length(typically downstream of both the BGE reservoir 20 and the samplereservoir 30), as shown in FIGS. 2G, 3E, pressure is decreased orreleased from the sample reservoir 30, but pressure is kept on at theBGE reservoir 20. As shown by the arrows, flow from the BGE channel 21clears sample in the sample and waste channels 31, 32, leaving a definedplug of sample Sp (trailing end is separated from any adjacent sample inthe cross channels 31, 32) in the primary fluid transport (e.g.,separation) channel 25. This pressure drive/injection is carried outwithout requiring any voltage input to the sample reservoir 30. At thispoint, as shown in FIGS. 2H, 3E, the pressure is released from the BGEreservoir 20 and voltage is applied between the BGE reservoir 20 and theseparation channel 25 at a downstream location, typically an end portionor terminus of the separation channel 25 such as at EO input 40 toperform an electrophoretic separation.

The voltage applied to the BGE reservoir 20 can be a high voltage HV,although lower voltages may be used in some embodiments. The voltage Vapplied downstream can be a lower voltage than the voltage applied tothe BGE reservoir 20. The lower voltage V can be any suitable EK drivingvoltage and may be between 10%-50% of the BGE reservoir voltage. Voltagecan vary and typically ranges from about +1 kV to +30 kV and the lowervoltage might range from 0 to +4 kV. But, the voltages and polarity canvary for different applications. For example, the polarity of theseparation could be reversed so that the high voltage input shown inFIG. 2H is negative, or closer to zero (0) and the opposing voltage(shown in FIG. 2H as the “+V” input) could be higher or even negativedepending on the relative length of the microfluidic channels, thecharge of the analytes, and the polarity of the ESI process. Embodimentsof the application refer to voltages that are referenced to a commonground or reference voltage with a value designated as “0” volts (“V”).

In some embodiments, each of the sample reservoir 30, the BGE reservoir20, and the waste reservoir 35 can be maintained at a common electricalpotential as the sample is flowed through the sample channel using onlypressure-driven operation so as to not apply an electrokinetic voltage,since these reservoirs are at the same electrical potential in theabsence of an external field. The sample can be introduced into theseparation channel 25 without applying an electrokinetic voltage and/orvoltage gradient across the sample channel 31 and/or SPE bed 28.

The sample can be flowed through the sample channel 31 without applyinga voltage to the sample reservoir 30, to the BGE reservoir 20, or to thewaste reservoir 35 and/or with no electric potential gradient in any ofthe sample channel 31, the BGE channel 21 and the waste channel 32.

The pressures applied to the headspaces of the reservoirs 20, 30 can below pressures, typically between 0.1 psi and 50 psi, more typicallybetween 0.5 and 30 psi, or between about 0.5 and about 12 psi or 10 psi(0.69 bars). The pressures can be about 0.5 psi, about 1 psi, about 1.5psi, about 2 psi, about 2.5 psi, about 3 psi, about 3.5 psi, about 4psi, about 4.5 psi, about 5 psi, about 5.5 psi, about 6 psi, about 6.5psi, about 7 psi, about 8 psi, about 8.5 psi, about 9 psi, about 9.5psi, about 10 psi, about 10.5 psi, about 11 psi, about 11.5 psi, about12 psi.

The reduced pressure such as a vacuum pressure applied to the wastereservoir 35 can be between about 1 psi and about 15 psi, typicallybetween about 10 psi and 15 psi, such as 10 psi, 11 psi, 12 psi, 13 psi,14 psi and 15 psi.

As noted above, tITP has been previously described as an online samplefocusing method for capillary electrophoresis. This phenomenon workswhen the sample contains a relatively large concentration of anelectrolyte (termed the leading electrolyte) that has higherelectrophoretic mobility than the analyte ions. As is well known, theleading electrolyte is typically added to the sample solution. Theleading electrolyte concentration should be significantly greater (suchas at least 5× or 10× greater) than the electrolyte concentration in thebackground electrolyte to provide a sufficient minimum conductivitydifference between the background electrolyte and the leadingelectrolyte. This is the situation that exists for the pressure-driveninjection of samples with high concentration of sodium chloride or otherdefined electrolyte. For example, for a pH 2.2 background electrolytewith a hydronium concentration of approximately 6 mM, a 15 mM leadingelectrolyte is too low, but concentrations at or above 50 mM aresufficient for tITP to be observed.

To take advantage of the sample focusing effects of tITP one can injecta larger band of this sample relative to other sampleprocessing/analysis methods and may use a suitable sample formulationwith the large concentration of the electrolyte. Where used, thepressure-driven injection methods allow increased if not total orcomplete freedom in altering the size of the sample band, simply bychanging the head pressure and/or the duration of the applied pressurefor the sample loading step. The BGE reservoir 20 can include liquidelectrolyte comprising sodium and/or salt in sufficient amount for tITPintroduced through channel 21. The liquid electrolyte may optionallyadditionally or alternatively be introduced via another reservoir andchannel, such as, via sample channel 31 or another side channel. ThistITP can be carried out before the separation shown in FIGS. 2H, 3F toreduce band broadening that may otherwise be introduced during thetransfer of the analyte sample from the SPE bed 28 to the separationchannel 25.

FIGS. 4A-4F and 6A are non-limiting examples of microfluidic devices 10that can be operated as described above. FIG. 4A illustrates the fluidicchannel 31 can include a plurality of spatially separated SPE beds 28,shown as first and second SPE beds 28 ₁, 28 ₂. Where more than one SPEbed 28 is used they can comprise the same or different sorbents and havethe same or different volumes and/or lengths.

FIG. 4B illustrates the microfluidic device 10 does not require a wastechannel 32 or waste reservoir 35. FIG. 4C illustrates the waste channel32 offset a longitudinal distance from the sample channel 31 across theseparation channel 25. FIG. 4D illustrates the device 10 can have aplurality of sample reservoirs 30, shown by way of example as three, 30₁, 30 ₂, 30 ₃, all or some of which can have the SPE bed 28, but more orless than three may be used. The reservoirs 30 can feed a common ordifferent sample channels 31, shown as having different sample channels31 ₁, 31 ₂, 31 ₃ all for a single separation channel 25, and at leastone BGE reservoir 20 (shown as a single BGE reservoir 20 and reservoirchannel 21). The sample reservoirs 30 can be controlled to sequentiallyor serially be pressure-driven (or, in some embodiments, EK driven) toinject respective sample plugs across the SPE bed 28 and into theseparation channel 25. The devices 10 can also optionally have an EOpump 40.

FIG. 4E illustrates a microfluidic device 10 that can have a pluralityof separation channels 25, shown as two adjacent channels, but more thantwo may be included on a single device 10. The separation channels 25can feed a common or separate emitters 50. Thus, the microfluidic device10 can include more than one separation channel 25 and associated BGEreservoir 20, reservoir 30, SPE bed 28, waste reservoir 35 and channels31, 32. One or more of the individual channels 21, 25, 31, 32 might beconfigured to have lateral dimensions of about 1-100 μm, e.g., about 75μm, with lateral spaced apart dimensions of about 1-100 mm, in someparticular embodiments.

As shown in FIG. 4F, one or more reference channels for a referencespray may be included on/in the microfluidic device 10. Where used, thereference material for the reference spray from a respective ESI emitter50 can provide one or more ions for internal calibration. In someembodiments, the reference material provides a single defined ion forinternal calibration. In other embodiments, the reference material caninclude multiple ions over a desired range, typically that are oversubstantially an entire m/z range of interest, to improve the massaccuracy.

FIG. 5A is a schematic illustration of a BGE reservoir 20 having agas-tight fitting holding a pressure supply line 70 and a (typicallyhigh) voltage line 75 with a high voltage input 75 i (shown as aplatinum wire) that extends inside the sealed reservoir 20 so as to beable to make contact with the fluid, e.g., liquid, in the reservoir 20.The term “gas-tight” means that the seal on the reservoir 20, 30 doesnot unduly leak when operated so as to be able to provide the desiredpressure to the headspace 20 h, 30 h, 35 h for pressure-driveninjection.

The pressure supply line 70 can be provided with tubing with an openpressurized gas path extending into the sealed headspace 20 h. For anexample of an 8 mm inner diameter reservoir wall 20 w, the pressuresupply line can be tubing less than this size, e.g., ¼ inch to about1/16 inch outer diameter. However, larger size conduits can be used whenstepped down in size for the supply into the reservoir head space underthe sealed connection. The sealed (e.g., gas-tight) connection of arespective pressurized gas supply line 70 to either reservoir 20, 30(and/or waste reservoir 35) can be provided via epoxy, O-ring, metal orelastomeric gaskets, grease fittings, and/or other suitableconfigurations. FIG. 5B illustrates that the pressured gas supply line70 can be held adjacent the high voltage cable 75 in a common sleeve 80.It is further noted that the high voltage cable 75 can be held routedinto the headspace while held inside the gas supply tubing.

FIG. 5B also illustrates that the top of the reservoir 20 t can besealed with a cap 20 c and a side port 20 p can be used to attach thepressurized gas supply line 70 to the reservoir 20. The same arrangementcan be used for the sample reservoir 30 and/or waste reservoir 35 (notshown). In some embodiments, the gas supply line 70 can be attached overthe outer wall of the wall 20 w of the reservoir 20 instead of extendinginside the reservoir 20 for gas-tight or sealed connection. Otherconnection configurations may be used.

FIG. 7A illustrates an example of a microfluidic system 100 whichincludes a controller 100 c used to control operation of the pressuresupply to the reservoirs 20, 30 to carry out a pressure-driveninjection. The system 100 can include first and second pressurized gassupply lines or conduits 70, shown as 70 ₁, 70 ₂, each in fluidcommunication with at least one valve 120, 130. The system 100 caninclude a three-way valve (FIG. 7B) that closes and opens each supplyline of the valves 120, 130. However, in preferred embodiments, separatevalves 120, 130, 135 are used for each supply line 70 ₁, 70 ₂ (and 70₃). One or all of the valves 120, 130, 135 can be a three-way valve(e.g., three way operation, open/close to source, open/close to headspace and open/close to atmosphere) for a respective supply line 70which can allow for the rapid venting of pressurized gas from arespective supply line. Thus, in operation, one or both of the valves120, 130, 135 can be operated to vent the head pressure in the reservoir20, 30, to atmosphere, which may help precisely control the injectionprocess. One or both of the gas supply lines 70 and/or reservoirs 20,30, 35 can also or alternatively include vents 121, 131 (FIG. 7A) thatcan be electronically opened and closed, for rapid venting to atmosphereto decrease pressure in a respective headspace 20 h, 30 h. The term“rapid” with respect to the venting or pressure reduction (e.g., ventingto atmosphere) in a respective pressure supply line 70 refers to a dropin pressure of the corresponding headspace 20 h, 30 h, 35 h of arespective reservoir 20, 30 and 35 to at least atmospheric pressurewithin 0.1-3 seconds, more typically within about 2 seconds or withinabout 1 second. The rapid venting can be based on a control signal fromthe controller 100 c that (a) directs the valve 120 or 130 or 135 toopen to atmosphere (where a three-way valve is used) or (b) opens a ventseparate from the valve 120, 130, 135 and closes the valve 120, 130,135. The rapid pressure reduction (e.g., venting) can be measured by apressure sensor in the supply line or reservoir to indicate the rapiddrop in head pressure from an operative pressure to atmospheric pressurewithin a 0.1-2 second time period. In some embodiments, the rapidventing can be carried out in between about 0.1 seconds and 1.5 seconds,such as about 0.1 seconds, about 0.2 seconds, about 0.3 seconds, about0.4 seconds, about 0.5 seconds, about 0.6 seconds, about 0.7 seconds,about 0.8 seconds, about 0.9 seconds, about 1 second, about 1.1 seconds,about 1.2 seconds, about 1.25 seconds, about 1.5 seconds, about 2seconds, and about 2.5 seconds.

The first and second pressurized gas supply lines 70 ₁, 70 ₂ can each bein communication with a common pressurized gas source 90 or each mayhave its own pressurized gas source. The system 100 can include a powersupply 95 for the high voltage input to the microfluidic device 10. Thepower supply 95 can be attached to the cable 75.

The controller 100 c can direct the timing sequence of thedifferentially applied pressure to the microfluidic device. Thecontroller 100 c can be in communication with the valves 120, 130, 135the at least one pressure source 90 (and a pressure reducing device 91,where used) and the power supply 95. The term “controller” is usedbroadly to include a single or multiple processors or applicationspecific integrated circuit (ASIC) held on or in communication with asingle device, e.g., the microfluidic device 10, and/or computer,laptop, notebook, smartphone and the like, or distributed in differentdevices using wires or wireless connections including local areanetworks or wide area networks, e.g., the internet, including any serversystem.

The controller 100 c can direct the valves 120, 130, 135 to open andclose to carry out successive sample injections using a definedsequence, an example of which is shown in the timing chart of FIG. 8. Inthe example shown, the SPE can be packed before the sample is loaded.The packing can be carried out using a reduced pressure (relative topressure applied to the other reservoirs) such as a vacuum V1 applied tothe waste reservoir 35 with or without pressure applied to either thesample channel 31 or BGE channel 21, typically without such otherpressures. The packing of the SPE bed can be carried out using a definedpressure for a defined time, typically between 1-10 minutes. PressuresP1, V1 can be applied for a defined time that is typically less than thepacking duration. The sample can be loaded using the pressures P1, V1,typically for a duration that less than the packing time, such asbetween 30-50% of the packing time and/or between 1-8 minutes. PressureP2 can then be applied concurrent with pressure P1 to inject the sampleplug into the separation channel 25. Then, the electric field can beapplied to separate the sample plug and spray the sample toward an inletof an MS. The broken line shown for P2 illustrates that this pressurecan be ON (typically at the same or a lesser amount as for theseparation pressure).

The SPE bed can be pre-conditioned for a short time, typically betweenabout 10 seconds to about 1 minute before the loading of the sampleusing V1 and P1 as shown. The elution can include applying a shortduration (i.e., 0.1-1 second) of pressures V1, P1 followed by a longerduration of pressures P1 and P2 for the injection/sample plug deliveryto the separation channel V1 may be OFF or ON during the elutiondelivery.

It is noted that the electrophoretic separation voltage can be appliedconcurrently with or just after pressure P2 is removed or decreased fromthe BGE reservoir 20. As shown, sample injection is carried out usingonly pressure P2 applied to the BGE reservoir and only pressure P1applied to the sample reservoir from the first and second supply lines70 (e.g., tubes or conduits) without any electrokinetic (EK) voltage.Voltage can be applied to the BGE reservoir 20 after the injection forthe ESI emitter spray 50 s.

The controller 100 c can be configured to operate the fluidic device 10using a defined timing sequence for applying defined pressures(headspace pressures) between 0.1 and 30 psi to a headspace 20 h of theBGE reservoir 20 via the supply tube 70 ₂ and to a headspace 30 h of thesample reservoir 30 via the supply tube 70 ₁ for defined durations,typically between 1 and 10 seconds, to inject a sample into theseparation channel 25. The timing chart shown in FIG. 8 is by way ofexample and the noted “zero” pressures of P1 (for the sample reservoir30) and P2 (for the BGE reservoir 20) may be atmospheric or ambientpressures or may alternatively be vacuum pressures. The applied voltageV from the power supply 95 to the input 75 i in the BGE reservoir 20(bottom line of the timing chart in FIG. 8) can have a shorter or longerduration than the concurrent injection pressures P1, P2 (FIG. 2F) or thesubsequent “clearing” pressure P2 (FIG. 2G). The P2 pressure can remainconstant or change, typically decreasing, from the concurrent pressurefor injection to the “clearing” pressure when P1 is “OFF” orsubstantially decreased (FIG. 2G).

The fluidic device 10 can be a microfluidic chip that has a substrate 10s and/or ceiling 10 c that is formed of hard or substantially rigidmaterials that include, but are not limited to, substrates comprisingone or combinations of: glass, quartz, silicon, ceramic, siliconnitride, polycarbonate, and polymethylmethacrylate. In particularembodiments, the device 10 can include a glass substrate such as aborosilicate. In other embodiments, a rigid polymer material may be usedto form the microfluidic device. The device 10 can also include one ormore layers of a soft or flexible substrate. Soft substrate materials,where used, can have a low Young's Modulus value. For example,elastomers and harder plastics and/or polymers can have a range betweenabout 0.1-3000 MPa. Examples of soft materials include, but are notlimited to, polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA),and polyurethane. See, e.g., co-pending PCT/US2012/027662 filed Mar. 5,2012 and PCT/US2011/052127 filed Sep. 19, 2011 for a description ofexamples of microfabricated fluidic devices. See, also, Mellors, J. S.;Gorbounov, V.; Ramsey, R. S.; Ramsey, J. M., Fully integrated glassmicrofluidic device for performing high-efficiency capillaryelectrophoresis and electrospray ionization mass spectrometry. Anal Chem2008, 80 (18), 6881-6887. Additional aspects of such devices aredisclosed, for example, in Xue Q, Foret F, Dunayevskiy Y M, Zavracky PM, McGruer N E & Karger B L (1997), Multichannel Microchip ElectrosprayMass Spectrometry. Anal Chem 69, 426-430, Ramsey R S & Ramsey J M(1997), Generating Electrospray from Microchip Devices UsingElectroosmotic Pumping. Anal Chem 69, 1174-1178, Chambers A G, Mellors JS, Henley W H & Ramsey J M (2011), Monolithic Integration ofTwo-Dimensional Liquid Chromatography—Capillary Electrophoresis andElectrospray Ionization on a Microfluidic Device. Analytical Chemistry83, 842-849. The contents of these documents are hereby incorporated byreference as if recited in full herein.

EO pumps can be integrated on a microfluidic device 10 for electrosprayionization via implementations other than the examples shown in FIGS.4A-4F. In general, two channels intersect at a junction, which may be aT-like junction (not restricted to a right angle intersection). Avoltage is applied to two of the three resulting channel terminigenerating an axial electric field through the associated channelsegments. To realize hydraulic transport through the third channelsegment, the electroosmotic mobility in the two channel segments thatcontain the axial electric field is generally different in magnitudeand/or sign. The difference in electroosmotic mobility can be achievedby chemically modifying one, or both, of the associated channel segmentsso as to produce different surface charge densities and hence differentelectroosmotic mobilities. Electroosmotic mobility can also be modifiedby coating a channel wall with electrically neutral polymer films,thereby increasing the effective fluid viscosity within the electricaldouble layer at the wall. Another way to modify electroosmotic mobilityis reduce one of the channel lateral dimensions to distances similar inmagnitude to the Debye length of the solution being electroosmoticallypumped. The described methods for modifying electroosmotic mobility mayalso be used in combination where desired. Methods for electroosmoticpumping are further described in U.S. Pat. No. 6,110,343, the contentsof which are hereby incorporated by reference.

While it is convenient to monolithically integrate EO pump functionalelements on electrospray microfluidic devices, it is possible tohydraulically deliver sample materials to the emitter. See, e.g.,Chambers A G, Mellors J S, Henley W H & Ramsey J M (2011) MonolithicIntegration of Two-Dimensional Liquid Chromatography—CapillaryElectrophoresis and Electrospray Ionization on a Microfluidic Device.Analytical Chemistry 83, 842-849. When utilizing hydraulic transport tosupply analyte to the emitter, electrical connection for producing theelectrospray, voltage can be achieved using a side channel similar tothe EO pumping channel or by contacting the fluid using an electrode ina reservoir external to the microfluidic device, or in the case of usingmetal tubing between the device 10 and the pump, connection can be madeto the tubing.

FIGS. 9A and 9B schematically illustrate embodiments of the invention.FIG. 9A illustrates a portable mass spectrometer 200 with a housing 201holding at least one of the microfluidic devices 10 with an onboardcontroller 100 c, a power supply for the voltage 95, a pressurized gassupply 90, a pressure reducing device 91, a detector 205, an analyzer210 and an optional display 215 for providing output data.

FIG. 9B illustrates that the microfluidic device 10 can be incommunication with a mass spectrometer 200 with an MS input 200 i facingthe ESI emitter 50 for receiving the spray 50 s. The controller 100 ccan be separate or partially or totally onboard the mass spectrometer200.

FIG. 10A is a flow chart of exemplary operations that can be used tocarry out a sample analysis. A fluidic device is provided (block 270).The fluidic device has at least one separation channel, at least one BGEchannel in fluid communication with the separation channel, at least onewaste channel in fluid communication with the separation channel and atleast one sample channel in fluid communication with the separationchannel.

The sample channel can have a pre-formed SPE bed therein.

The SPE bed can be flowably formed in the sample channel (block 272).

The SPE bed can be flowably formed by applying a reduced pressure via apressure reducing device to the waste channel while applying a greaterpressure to the sample channel to flow a slurry of SPE material into thesample channel until it hits at least one blocking member residingupstream of the separation channel to flowably form the SPE bed.

The SPE bed can be pre-conditioned by flowing solvent across the SPE bedand into the waste channel (block 275).

A low concentration of an ion-pairing agent such as TFA to retain asample can be introduced before introducing the sample per block 280 andthen the TFA can be removed prior to elution of block 285 (block 283).

The sample can be flowed over/through at least one SPE bed in the samplechannel toward the waste channel (block 280).

Wash solvent can be flowed across the at least one SPE bed toward thewaste channel after the same is flowed over the SPE bed (block 282).

Elution fluid can then be flowed over/through the at least one SPE bedtoward the waste channel to load a sample plug into the separationchannel (block 285).

BGE liquid can then be flowed into the separation channel a distancebelow/downstream of the waste and sample channels to clear theseparation channel and adjacent waste and sample channels while movingthe sample plug downstream of the waste and sample channels in theseparation channel (block 288).

tITP can be performed (using the BGE reservoir) prior to the CEseparation of block 288 to narrow the injection band and reduce bandbroadening otherwise associated with transfer of the sample plug/bandfrom the SPE bed to the separation channel (block 289).

An electric field can be applied to the separation channel to separateadjacent plugs, forcing the sample plug to travel toward an ESI emitterand cause the ESI emitter to spray the sample (block 290).

The sprayed sample can be analyzed in a mass spectrometer to determineinformation about the sample (block 295). The analysis can includedetecting analyte peak signals of the sample using a mass spectrometerand generating electropherograms of the sample, for example.

Electronic detection of signal of the separated sample in the separationchannel can be obtained using a detector in communication with theseparation channel (optically and/or electronically). This electronicdetection can be carried out without the mass spectrometer detection orwith the mass spectrometer detection. In some embodiments, theelectronic detection by the detector is carried out simultaneously withdetection by the mass spectrometer for a respective separated sample.

The pre-conditioning, flowing and loading steps can be carried out usingonly a sequence of defined pressure inputs to the BGE channel, thesample channel and the waste channel without requiring electric fieldsuntil the separation and spray.

FIG. 10B is a flow chart of exemplary operations that can be used tocarry out a sample analysis. A microfluidic device is provided with atleast one separation channel in fluid communication with a backgroundelectrolyte (BGE) reservoir and a sample reservoir and having a samplechannel with an SPE bed; the sample channel merges into the separationchannel (block 300). A fluid sample is injected (flowably introduced)from the sample reservoir across the SPE bed into the separation channeldownstream of the BGE reservoir by concurrently applying a definedpressure to the BGE reservoir and a defined pressure to the samplereservoir (block 310).

A reduced pressure (optionally a vacuum) can be applied to a wastereservoir residing across from the sample channel during the injectingstep.

After the injection/introduction, a trailing end of the sample plug iscleared from the sample channel using fluid flowed from the BGEreservoir to deliver the sample plug in the separation channel inresponse to reducing or removing the pressure applied to the samplereservoir while applying pressure to the BGE reservoir so that pressureapplied to the BGE reservoir is greater than pressure then applied tothe sample reservoir (block 320). Then, the delivered sample iselectrophoretically separated in the separation channel by applying anelectric field to the separation channel, i.e., voltage to the BGEreservoir and a downstream location of the separation channel orproximate EO pump channel/input (block 330). The pressure in the BGEreservoir can be held constant, further reduced or removed while thevoltage is applied.

The injecting, clearing and electrophoretic separation can be carriedout without applying a voltage to the sample reservoir (block 340).

A duration of the pressure can be electronically adjusted or thepressure applied to the sample or BGE reservoir 20, 30 can be increasedor decreased for the injecting and/or clearing to adjust a size of theplug of the sample delivered to the separation channel.

As the analyte bands elute from the distal end of the electrophoreticseparation channel, the separated sample can be analyzed, optionallyusing a mass spectrometer to determine information about the sample(block 335). The analysis can include detecting analyte peak signals ofthe sample using a mass spectrometer and generating electropherograms ofthe sample, for example. The analysis can comprise electricalconductance detection and/or optical detection of the separated samplein the separation channel to obtain quantitative and/or qualitative dataof the analytes in the sample.

It is noted that embodiments of the present invention may combinesoftware, firmware and/or hardware aspects, all generally referred toherein as a “circuit” or “module.” Furthermore, the present inventionmay take the form of a computer program product on a computer-usablestorage medium having computer-usable program code embodied in themedium. Any suitable computer readable medium may be utilized includinghard disks, CD-ROMs, optical storage devices, a transmission media suchas those supporting the Internet or an intranet, or magnetic storagedevices. Some circuits, modules or routines may be written in assemblylanguage or even micro-code to enhance performance and/or memory usage.It will be further appreciated that the functionality of any or all ofthe program modules may also be implemented using discrete hardwarecomponents, one or more application specific integrated circuits(ASICs), or a programmed digital signal processor or microcontroller.Embodiments of the present invention are not limited to a particularprogramming language.

Computer program code for carrying out operations of the presentinvention may be written in an object oriented programming language suchas Java®, Smalltalk or C++. However, the computer program code forcarrying out operations of the present invention may also be written inconventional procedural programming languages, such as the “C”programming language. The program code may execute entirely on theuser's computer, partly on the user's computer, as a stand-alonesoftware package, partly on the user's computer and partly on anothercomputer, local and/or remote or entirely on the other local or remotecomputer. In the latter scenario, the other local or remote computer maybe connected to the user's computer through a local area network (LAN)or a wide area network (WAN), or the connection may be made to anexternal computer (for example, through the Internet using an InternetService Provider).

Embodiments of the present invention are described herein, in part, withreference to flowchart illustrations and/or block diagrams of methods,apparatus (systems) and computer program products according toembodiments of the invention. It will be understood that each block ofthe flowchart illustrations and/or block diagrams, and combinations ofblocks in the flowchart illustrations and/or block diagrams, can beimplemented by computer program instructions. These computer programinstructions may be provided to a processor of a general purposecomputer, special purpose computer, or other programmable dataprocessing apparatus to produce a machine, such that the instructions,which execute via the processor of the computer or other programmabledata processing apparatus, create means for implementing thefunctions/acts specified in the flowchart and/or block diagram block orblocks.

These computer program instructions may also be stored in acomputer-readable memory that can direct a computer or otherprogrammable data processing apparatus to function in a particularmanner, such that the instructions stored in the computer-readablememory produce an article of manufacture including instruction meanswhich implement the function/act specified in the flowchart and/or blockdiagram block or blocks.

The computer program instructions may also be loaded onto a computer orother programmable data processing apparatus to cause a series ofoperational steps to be performed on the computer or other programmableapparatus to produce a computer implemented process such that theinstructions which execute on the computer or other programmableapparatus provide steps for implementing some or all of thefunctions/acts specified in the flowchart and/or block diagram block orblocks.

The flowcharts and block diagrams of certain of the figures hereinillustrate exemplary architecture, functionality, and operation ofpossible implementations of embodiments of the present invention. Inthis regard, each block in the flow charts or block diagrams representsa module, segment, or portion of code, which comprises one or moreexecutable instructions for implementing the specified logicalfunction(s). It should also be noted that in some alternativeimplementations, the functions noted in the blocks may occur out of theorder noted in the figures. For example, two blocks shown in successionmay in fact be executed substantially concurrently or the blocks maysometimes be executed in the reverse order or two or more blocks may becombined, or a block divided and performed separately, depending uponthe functionality involved.

FIG. 11 is a schematic illustration of a circuit or data processingsystem 290. The system 290 can be used with microfluidic devices 10and/or mass spectrometers 200. The circuits and/or data processingsystems 290 may be incorporated in a digital signal processor in anysuitable device or devices. As shown in FIG. 11, the processor 410 cancommunicate with a mass spectrometer 200 and/or microfluidic device 10and with memory 414 via an address/data bus 448. The processor 410 canreside in a control circuit or controller that is separate from thespectrometer 200 or that is integrated wholly or partially therein. Theprocessor 410 can be any commercially available or custommicroprocessor. The memory 414 is representative of the overallhierarchy of memory devices containing the software and data used toimplement the functionality of the data processing system. The memory414 can include, but is not limited to, the following types of devices:cache, ROM, PROM, EPROM, EEPROM, flash memory, SRAM, and DRAM.

FIG. 11 illustrates that the memory 414 may include several categoriesof software and data used in the data processing system: the operatingsystem 452; the application programs 454; the input/output (I/O) devicedrivers 458; and data 455. The data 455 can include sample type, plugsize adjustments for pressures, calibration data, time synchronizationdata (e.g., pressures/duration for loading/injection), and/or otherdetected or internal mass spectrometer data.

As will be appreciated by those of skill in the art, the operatingsystems 452 may be any operating system suitable for use with a dataprocessing system, such as OS/2, AIX, DOS, OS/390 or System390 fromInternational Business Machines Corporation, Armonk, N.Y., Windows CE,Windows NT, Windows95, Windows98, Windows2000, WindowsXP or otherWindows versions from Microsoft Corporation, Redmond, Wash., Unix orLinux or FreeBSD, Palm OS from Palm, Inc., Mac OS from Apple Computer,LabView, or proprietary operating systems. The I/O device drivers 458typically include software routines accessed through the operatingsystem 452 by the application programs 454 to communicate with devicessuch as I/O data port(s), data storage 455 and certain memory 414components. The application programs 454 are illustrative of theprograms that implement the various features of the data (image)processing system and can include at least one application, whichsupports operations according to embodiments of the present invention.Finally, the data 455 represents the static and dynamic data used by theapplication programs 454, the operating system 452, the I/O devicedrivers 458, and other software programs that may reside in the memory414.

While the present invention is illustrated, for example, with referenceto the Control Module for Microfluidic Devices with in-line SPE beds forCE-MS analysis of samples (typically with a Pressure Input Sequence) 450and an optional Plug Size (pressure/duration) Adjustment Module 451being application programs in FIG. 11, as will be appreciated by thoseof skill in the art, other configurations may also be utilized whilestill benefiting from the teachings of the present invention. The Module451 can allow for a user to select a desired injection time (Pressure ONtime, OFF time, pressure for a respective injection and/or clearing andthe like, for each reservoir). The Modules 450 and/or 451 may also beincorporated into the operating system 452, the I/O device drivers 458or other such logical division of the data processing system. Thus, thepresent invention should not be construed as limited to theconfiguration of FIG. 11, which is intended to encompass anyconfiguration capable of carrying out the operations described herein.Further, Module 450 and/or 451 can communicate with or be incorporatedtotally or partially in other components, such as a mass spectrometer200, power supply 95, an interface/gateway or a computer such as at aworkstation that may be local or remote from the microfluidicdevice/spectrometer.

The I/O data port can be used to transfer information between the dataprocessing system, the workstation, the spectrometer, the microfluidicdevice, the interface/gateway and another computer system or a network(e.g., the Internet) or to other devices or circuits controlled by theprocessor. These components may be conventional components such as thoseused in many conventional data processing systems, which may beconfigured in accordance with the present invention to operate asdescribed herein.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLES

LC-MS grade acetonitrile, methanol, acetone, and formic acid (99.99%)were acquired from Fisher Chemical (Fairlawn, N.J.) as well as HPLCgrade ammonium acetate and trifluoroacetic acid (TFA) (99.975%). Waterwas purified with a Nanopure Diamond water purifier (BarnsteadInternational, Dubuque, Iowa). (3-Amino)di-isopropylethoxysilane(APDIPES) was acquired from Gelest (Morrisville, Pa.). Sodium phosphatedibasic, trichloro(1H,1H,2H,2H-perfluorooctyl)silane was acquired fromSigma-Aldrich (St. Louis, Mo.). N-hydroxylsuccinimide functionalizedpolyethylene glycol (NHS-PEG) reagent with 450 polymer units (MW=20 kDa)was purchased from Nanocs, Inc. (Boston, Mass.). Met-enkephalin,bradykinin, angiotensin II, thymopentin, and human glu-fibrionpeptidewere purchased from American Peptide Company (Sunnyvale, Calif.).MassPREP Phosphorylase B and E. coli digest were procured from WatersCorporation (Milford, Mass.). A schematic for the SPE-CE-ESI microchipcan be found in FIG. 1A. The microchip was fabricated out of 550 μmthick D263 glass using standard photolithography, wet chemical etching,and thermal bonding techniques. See, Mellors et al., Anal Chem 2008, 80,6881-6887; and Mellors et al., Anal Chem 2010, 82, 967-973. All channelswere 10 μm deep and 70 μm wide (full-width). The microchip was thencoated with APDIPES and modified with an NHS-PEG reagent as describedpreviously. See, e.g., Batz et al., Anal Chem 2014, 86, 3493-3500; andRedman et al., Anal Chem 2015, 87, 2264-2272. The contents of thesedocuments are hereby incorporated by reference as if recited in fullherein.

To perform integrated sample processing, SPE-CE-ESI microchips werepacked with 5 μm diameter porous Oasis HLB particles provided by WatersCorporation (Milford, Mass.). The SPE bed was packed against amicrofabricated weir by placing a slurry of 0.1 mg/mL Oasis HLBparticles in acetone in reservoir 30 (FIG. 1A) and applying reducedpressure (optionally a vacuum) to reservoir 35 (FIG. 1A). The length ofthe packed bed was 600 μm long, while the channel depth and width were10 μm and 70 μm, respectively. The volume of the packed bed wasestimated to be 425 pL. The time required to pack the SPE bed on themicrochip was 10 minutes.

To perform SPE-CE-ESI, the bed was first conditioned with low organicsolvent. A 95:5 0.1% formic acid in water:methanol (v/v) solution wasplaced in reservoir 30 (FIG. 1A). Head pressure (+0.69 bar) from a tankof compressed nitrogen was applied to reservoir 30 while simultaneouslyapplying a vacuum to reservoir 35 for 30 seconds. To load sample ontothe SPE bed, the desired sample was placed in reservoir 30. Unlessotherwise noted, the sample was dissolved in 2:98 methanol:0.5% TFA inwater (v/v). The same pressure and vacuum were applied to the sample andwaste reservoir, respectively, for 5 minutes. Finally, elution solventreplaced the sample in reservoir 2. For SPE-CE-ESI, the elution solventwas 80:20 acetonitrile:2% formic acid in water (v/v).

For SPE-tITP-CE-ESI, the elution solvent was the same with the additionof 100 mM ammonium acetate. To perform an elution, the procedure was thesame for both the SPE-CE-ESI and SPE-tITP-CE-ESI methods. First, +0.69bar was applied to reservoir 30 for one second. Next, +0.69 bar wasapplied to both reservoirs 20 and 30 for four seconds. This resulted inthe eluted analyte band entering the separation channel. Next, +0.69 barwas applied to reservoir one (20), which cleared the injection cross ofexcess sample prior to the application of voltage and creation of theinjection plug. Finally, voltage was applied to reservoirs 20 (+22 kV)and 40 (+1.5 kV). The applied voltages resulted in a field strength of760 V/cm. The application of the pressure and vacuum to the chip wascontrolled using a 3-way electronic valve (Clippard, Inc., Cincinnati,Ohio), which was operated by an SCB-68 breakout box connected to a PCvia a PCI 6713 DAQ card (National Instruments, Austin, Tex.) andcontrolled by a Labview program. The BGE for all SPE-CE-ESI andSPE-tITP-CE-ESI separations was 50:50 acetonitrile:2% formic acid inwater, pH 2.2.

Separation of a Four Peptide Mix

A four peptide mix (thymopentin, bradykinin, angiotensin II andmet-enkephalin) was used to compare figures of merit for the threedifferent methods investigated. A Waters Synapt G2 quadrupoletime-of-flight (qTOF) mass spectrometer was used (Waters Corporation,Milford Mass.). The instrument was set to MS Sensitivity mode with amass range of 300-600 m/z and a summed scan time of 0.09 seconds.

Electrokinetic and Hydrodynamic Injection Comparison

To compare differences in observed peak area between an electrokineticgated injection and a hydrodynamic injection, microchip CE-ESI was usedto separate a 5 μM four peptide mix (thymopentin, bradykinin,angiotensin II and met-enkephalin). A 23 cm CE-ESI microchip wasutilized. The design is very similar to the microchip described in FIG.1A, however, no weir was present in the channel 31 connected toreservoir 30, and no SPE bed was present. The depth and the width of thechannels was the same as the SPE-CE-ESI microchip, as well as thesurface coating. The BGE and MS settings were the same as thosedescribed earlier. For the electrokinetic injection, the appliedvoltages were 22, 22, 20, and 2 kV, respectively. The duration of thegated injection was 0.4 s. For the hydrodynamic injection, 0.165 bar ofhead pressure was applied to reservoirs 20 and 30 simultaneously for 3seconds. This resulted in sample entering the separation channel. Next,0.165 bar of pressure was applied to reservoir 20 for one second, whichcleared the injection cross of excess sample creating the injection plugin the separation channel. Finally, applied voltages of 22 kV and 2 kVwere applied to reservoirs 20 and 40, respectively. The analyte volumesfor the injections were calculated by multiplying the volumetric flowrate (linear velocity x cross sectional area) and the injection time.

Sample Carry Over

Sample carry over was investigated for both the CE-ESI microchip and theSPE-CE-ESI microchip. For both techniques, a sample of the four peptidemix was separated at either 5 μM or 50 nM, respectively. Directlyfollowing the separation, the reservoir that contained the sample wasrinsed by removing the sample and filling the reservoir with BGE. Thesolution was mixed by aspirating with a pipette. This process wasrepeated three times and then the reservoir was filled with a blank. Forthe CE-ESI microchip, the blank consisted of BGE. For the SPE-CE-ESImicrochip, the blank consisted of 2% methanol, 0.5% trifluoracetic acid,and 97.5% water. For both techniques, the same sample loading andinjection procedures that were used for the peptide mix was applied tothe blank. The mass spectrometer settings were the same as thosedescribed for the separation of the 4-peptide mix.

Protein Digest Separations

A tryptic digest of Phosphorylase B was separated using both CE-ESI (5μM) and SPE-tITP-CE-ESI (50 nM). Additionally, 0.5 mg/mL E. coli digestwas analyzed using SPE-tITP-CE-ESI. A Waters Synapt G2 qTOF was used toperform MS^(E). The term “MS^(E)” refers to a single analyticaltechnique for Mass Spectrometers from Waters Corporation (Milford,Mass., USA) that can separate components of a complex sample andinterrogate quantitative and qualitative information for review. See,White Paper, An Overview of the Principles of MS^(E), the Engine thatDrives MS Performance, Copyright 2011, Waters Corporation. To be clear,this White Paper and the noted analytical technique are provided by wayof example only and the invention is not limited to this analysisprotocol or this equipment. The instrument was set to MS/MS Sensitivitymode with a mass range of 50-1200 m/z and a summed scan time of 0.05seconds. Human Glu-fibrinopeptide precursor was used as a lockmasscompound, as described earlier.

Data Processing

To determine peak width and area, extracted ion electropherograms wereexported into the program Igor Pro (Wavemetrics, Lake Oswego, Oreg.) andthe analysis package “multi-peak fit 2” was used. To determine the peakcapacity, base peak index (BPI) electropherograms were exported to theprogram PeakFinder (Pacific Northwest National Laboratory), whichmeasured the separation window and median 4σ peak width.

The Phosphorylase B digest electropherograms were analyzed using thesoftware Biopharmalynx (Waters Corporation) to determine the number ofpeptides observed. The method was based on a trypsin enzyme digest withup to two missed cleavages permitted. The MS and MS^(E) mass toleranceswere both set to 30.0 ppm. The MS ion intensity threshold was set to 25counts, while the MS^(E) ion intensity threshold was set to 10 counts.Only peptides with an intensity value greater than or equal to 1% of themost intense peptide were counted.

Results and Discussion

Separation and Pre-Concentration Performance

To investigate the concentration and separation performance of theintegrated sample processing microchip three different approaches werecompared; conventional CE-ESI, SPE-CE-ESI, and SPE-tITP-CE-ESI. Allmicrochips were coupled directly with MS for detection. To ensure theseparation conditions were identical for all methods, the CE separationswere performed using the SPE-CE-ESI device without any stationary phase.Microchip CE-ESI separations were performed using a gated electrokineticinjection, while the SPE-CE-ESI and SPE-tITP-CE-ESI methods used ahydrodynamic injection. As long as the injection volume is sufficientlylow to prevent injection broadening, the injection method will not haveany influence on the separation performance. The affect of the injectionmethod on the sensitivity of the methods is explored later. FIGS.12A-12C compare the electropherograms for the three techniques whileTable 1 compares the figures of merit. Theoretical plates are not anaccurate measure of separation efficiency when tITP is coupled with CE,as the field strength is not linear across the channel for the durationof the separation. Since peak capacity does not require a linearelectric field, it was therefore employed as a more accurate performancemetric.

FIGS. 12A-12C show representative electropherograms from the threetechniques. The peaks are labeled 1 through 4, and correspond tothymopentin, bradykinin, angiotensin II, and met-enkephalin,respectively. Peaks A (1087.5 Da) and B (1073.5 Da) correspond tounidentified trace peptides that were observed in the SPE-CE-ESI andSPE-tITP-CE-ESI electropherograms. The top electropherogram (FIG. 12A)corresponds to the CE-ESI analysis of a 5 μM peptide mix. The observedmigration times for the four peptides were 1.64, 1.84, 1.98, and 4.19min, respectively with a total separation time of just over four minuteslong. The efficiency values for the CE-ESI approach were between 90,000and 133,000 theoretical plates. The average peak capacity observed was105.5, with a median 4σ peak width of 1.45 seconds and a separationwindow of 152.95 s (n=3). By using these figures of merit as a baseline,we can compare the separation performance of the SPE-CE-ESI andSPE-tITP-CE-ESI methods.

The middle electropherogram (FIG. 12B) corresponds to the SPE-CE-ESIanalysis of 50 nM peptide mix, 100 times more dilute than the sampleused for the CE-ESI separation. The migration time of the four peptideswas 1.72, 1.92, 2.07 and 4.27 min respectively, very similar to themigration times for the CE-ESI electropherogram. Peak 5, at 4.50 mincorresponds to oxidized met-enkephalin, which was not visible in theCE-ESI electropherogram, indicating the concentration enhancement of theSPE-CE-ESI approach. The theoretical plate counts of the four peptidesusing the SPE-CE-ESI method were between 39,000 and 92,000 theoreticalplates and the observed peak capacity for the SPE-CE-ESI method wasdecreased to 66.5, with a median 4σ peak width of 2.30 s and aseparation window of 152.95 s (n=3) (Table 1). These values did not takethe oxidized met-enkephalin peak (peak 5) into account. FIG. 12C is aSPE-tITP-CE-ESI of a 50 nM sample.

TABLE 1 Calculated peak capacity and efficiency values for 4-peptide mixPeak Angiotensin Met- Capacity Thymopentin Bradykinin II EnkephalinCE-MS 105.5 107,526  90,583 109,349  132,782  SPE-CE-MS 66.5 39,06141,945 45,731 91,979 SPE-tITP-CE- 174.5 407,571* 311,383* 300,563*153,868* MS *Apparent efficiency values calculated based on peak widthand migration time

To investigate the amount of pre-concentration observed, an enrichmentfactor was calculated for each of the four peptides. To characterize theeffect of the different injection methods (hydrodynamic versuselectrokinetic), microchip CE-ESI of the same four peptides wasperformed using both injection methods. The volume of analyte injectedinto the separation channel for an electrokinetic gated injectiondepends on the electrophoretic mobility of the analyte. For ahydrodynamic injection, the volume should be constant for all analytes.When comparing the pre-concentration performance of the SPE-CE-ESI andSPE-tITP-CE-ESI methods, it may be important to distinguish if anyincrease in observed sensitivity is due to pre-concentration of analyteon the bed or due to the removal of electrokinetic bias. FIGS. 13A and13B illustrate the electropherograms for the electrokinetic (top, FIG.13A) and hydrodynamic (bottom, FIG. 13B) injections for the four peptidemix. The peptides are labeled 1 through 4, corresponding to thymopentin,bradykinin, angiotensin II, and met-enkephalin. For the electrokineticinjection, the injection volumes of peaks 1 through 4 were calculated tobe 585, 522, 482, and 215 pL, respectively. For the hydrodynamicinjection separation, the injection volume was matched to the largestvolume from the electrokinetic injection, and was calculated to be 600pL. As illustrated by the figure as well as the injection volumes, theelectrokinetic bias increases as the analyte migration time increases,corresponding to peptides with lower electrophoretic mobilities. Theobserved bias is especially prevalent for met-enkephalin (peak 4), whilethe average (n=3) peak heights for peaks 1 through 3 are within 20% ofeach other for each pair.

From the electropherograms in FIGS. 13A and 13B, the average (n=3replicate injections) peak areas were calculated and compared betweenthe two methods. FIG. 14 plots the calculated peak areas versusmigration time for each of the four peptides. As illustrated by FIG. 14,no significant difference in peak area was observed for the first threepeptides (peaks 1 through 3). However, a significant difference wasobserved for the met-enkephalin peak, the peptide with the lowestelectrophoretic mobility, and therefore the highest electrokinetic bias.To account for this difference in sensitivity, a correction factor canbe calculated by using a ratio of the electrokinetic injection peak areato the hydrodynamic injection. For met-enkephalin, this value wascalculated to be 0.31, which is similar to the calculated difference ininjection volume (215 v. 600 pL).

For the SPE-CE-ESI method, the enrichment values were 78, 712, 713, and799 for peaks 1-4, respectively (FIG. 12C). The enrichment factor wascalculated by multiplying the difference in initial analyteconcentration (100) by a ratio of the SPE-CE-ESI peak area compared tothe CE-ESI peak area. For the met-enkephalin peak, the correction factor(0.31) was also used to account for the difference in peak area due tothe differences in injection method.

The enrichment factor will vary due to differences in retention on theSPE bed as well. Thymopentin (Peak 1), a small (679.76 Da), hydrophilicpeptide was not well retained on the hydrophobic stationary phase andthus had a much lower enrichment factor compared to the other peptides.Without the addition of TFA (0.5%) to the sample, thymopentin was notretained at all on the stationary phase (data not shown). Therefore, thelower observed enrichment factor of thymopentin compared to the otherpeptides was expected. TFA is an ion-pairing agent commonly used inchromatography, however, it is typically incompatible with ESI due toion-suppression effects. The SPE-CE-ESI approach does not suffer thislimitation, as TFA can be employed to retain the sample but can then beremoved prior to elution. The ability to utilize TFA to improveretention of analytes on the SPE bed without compromising ESIperformance represents a significant advantage of the SPE-CE-ESImicrochip compared to LC-MS instruments where the TFA content isintroduced directly into the mobile phase. The enrichment factors forbradykinin and angiotensin II were approximately 700, while thecalculated value for met-enkephalin was 799. Overall, a significantamount of pre-concentration was observed using the SPE-CE method for thepeptide mix. However, the resulting separation performance was reducedto roughly half that of the CE-ESI method, highlighting the challenge ofcoupling sample processing with CE-ESI while maintaining separationperformance.

In order to regain the separation performance lost using the SPE-CE-ESImethod while maintaining the observed pre-concentration, tITP was usedprior to the CE-ESI separation. As tITP is a focusing technique, itproduced a narrow injection band, eliminating any band broadeningintroduced by the transfer of the analyte band from the SPE bed to theCE channel. The electropherogram in FIG. 12C corresponds to the analysisof 50 nM peptide mix using the SPE-tITP-CE-ESI method. The procedure forthis method is identical to the SPE-CE-ESI technique, except that 100 mMammonium acetate was added to the elution solvent. The ammonium acts asthe leading electrolyte and the formic acid in the BGE acts as thetrailing electrolyte. Directly after the application of voltage to themicrochip, the sample undergoes tITP focusing. Following the focusingstep, the sample transitions to a CE separation. The migration times forthe peptides are 1.68, 1.86, 1.98, and 3.98 min, respectively. Comparedto both the CE-ESI and SPE-CE-ESI electropherograms, the separationwindow was compressed in the SPE-tITP-CE-ESI electropherogram due to thetITP focusing step. The average peak capacity observed was 174.5, with amedian 4σ peak width of 0.81 s and a separation window of 141.33 s(n=3). This is nearly double compared to the CE-ESI separation, andnearly triple that for the SPE-CE-ESI method. The enrichment values forthe four peptides were calculated to be 72, 614, 660, and 783,respectively. As evident by both the calculated peak capacity andenrichment values, the separation performance for the SPE-tITP-CE methodis greatly improved compared to the CE and SPE-CE methods whileproviding a significant amount of sample pre-concentration.

Finally, to demonstrate the robustness of the developed methods themigration time reproducibility, peak area reproducibility, samplecarryover, and limits of detection were investigated. For threereplicate injections of the peptide mix, the average migration time RSDvalues were 0.23% for CE-ESI, 0.49% for SPE-CE-ESI, and 0.94% forSPE-tITP-CE-ESI. The average peak area RSD values were 7.08%, 5.26%, and8.66%, respectively. These values indicate that both the SPE-CE-ESI andSPE-tITP-CE-ESI methods are reproducible, with migration time and peakarea RSDs below 1 and 10%, respectively. To determine the limit ofdetection (LOD), the signal to noise (S/N) ratio of each peptide wascalculated and then extrapolated to a S/N of 3. For the CE-MS method,the LOD of peaks 1 through 4 were calculated to be 800, 700, 400, and700 nM, respectively. For the SPE-tITP-CE-MS method, the values were3.0, 2.2, 2.6, and 1.7 nM, respectively, indicating more than two ordersof magnitude lower LOD for the method with integrated sample processing.Sample carryover was compared for the SPE-CE and compared to the CEmethod. Following the separation of a four peptide mix (CE—5 μM,SPE-CE—50 nM), a blank was analyzed. Sample carryover was characterizedby observing detection of any peptides in the blank electropherogram.For the CE electropherograms, no sample carryover was detected. For theSPE-CE electropherogram, carryover was only observed for bradykinin, thepeptide with the most intense signal, yielding a peak area value of only0.04% of the initial peak. This sample carryover is considerednegligible and further optimization of wash steps between samples willlikely eliminate it completely. The sample carryover performance of theSPE-CE-ESI method can be extrapolated to the SPE-tITP-CE-ESI method. Thepresence of salt in the elution solvent is the only difference betweenthe two methods with all other steps identical, including the sampleloading step and a wash step of the SPE bed following elution.Therefore, any residual material left on the SPE bed should be identicalbetween the two methods. The high enrichment factors, low RSD values,low sample carryover and improvement in separation performancedemonstrate the viability of this method for analyte pre-concentrationfor CE-ESI.

Dynamic Range and Overloading

When employing a stationary phase to pre-concentrate samples it can beimportant to ensure that the SPE bed does not become saturated in thedesired concentration range. The integrated SPE-tITP-CE-MS methodpossesses three dimensions that are subject to sample overloading; theCE separation, the MS detector, and the adsorbent bed. As the sample canbe continually loaded onto the adsorbent (SPE) bed, both volume and massoverloading must be considered. Volume overload occurs when a largevolume (orders of magnitude larger than the volume of the stationaryphase bed) of sample is injected onto the bed. Even under solventconditions designed for sample adsorption (low organic), the largevolume can cause weakly retained analytes to partition into the mobilephase and be eluted from the bed. Mass overload occurs when the amountof analyte injected exceeds the capacity of the stationary phase,saturating the bed with sample. It can be difficult to assign theeffects of overloading to the saturation of the stationary phase aloneand experiments must be carefully designed to ensure that it ischaracterized appropriately.

In order to determine the applicable dynamic range for the SPE-tITP-CEmethod the four peptide mix was investigated. The concentration rangeexplored was 1, 10, 100, 1000 and 10000 nM. FIGS. 15A-15D illustrate BPIelectropherograms for the peptide mix between 10 nM and 10 μM. Thepeptides were not visible in the BPI electropherogram of the 1 nMsample, but were observed in the extracted ion electropherogram. Asdemonstrated by FIGS. 15A-15D, the peak height and area for the fourpeptides increased as the concentration increased. The peak shape becamedistorted at high concentrations, and it appears that the detectorbecomes saturated. Furthermore, trace impurities were also present inthe four peptide mix, and were observed at higher concentrations. Two ofthese impurities were labeled peptide A and B. The concentration ofpeptide A and B in the mixture were unknown, but it's estimated thatthey were at least an order of magnitude less concentrated than the foursample peptides.

FIGS. 16A-16C, 17A-17C and 18A-18C illustrate the peak area, width, andheight of bradykinin, thymopentin, and peptide A, respectively, over therange of concentrations tested. These plots illustrate peak area, width,and height for SPE-tITP-CE-ESI analysis of bradykinin, thymopentin, andan unidentified trace component labeled peptide A. The concentration ofthe peptide mix loaded ranged from 1 nM to 10 μM. Sample concentrationreflects the concentration of the 4 peptides in the mixture(thymopentin, bradykinin, angiotensin II, and met-enkephalin) and doesnot represent the concentration of the trace components A and B.

To determine overloading in the CE domain the peak width of bradykininand peptide A were compared. The peak width of bradykinin remainsunchanged from 1 nM to 100 nM. Above 100 nM, the peak width sharplyincreases. On the other hand, the peak width of the peptide A remainssimilar over the range of concentrations. This suggests that the CEseparation has been saturated for the well-retained, highly abundantanalyte. FIGS. 15A-15D visually support this conclusion. Monitoring thepeak heights of the three peptides assesses the saturation of the MSdetector. The peak height of bradykinin increased linearly (R²=0.999)from 1 nM to 100 nM. However, at concentrations of 1 and 10 μM, the peakheight deviated from the linear behavior and plateaued. The peak heightof the weakly retained thymopentin increased linearly (R²=0.999) between1 nM and 100 nM. At 1 μM, the height began to deviate from linear andsubsequently decreased. Comparatively, the peak height of the lowabundant peptide A is linear (R²=0.998) for all four concentrations ofsample detected. This indicates that the MS detector had been saturatedfor the highly abundant species.

The values for peak area and height of the thymopentin suggest thatvolume overloading of the stationary phase is occurring. This is notsurprising as the volume of sample is continually being introduced tothe short bed (600 μm) for 5 minutes. In addition, the capacity of thebed for weakly retained analytes may decrease as the presence of othercompounds with higher retention increases. Finally, to determine massoverload bradykinin and peptide A were compared. The peak area ofbradykinin is linear through the first four orders of magnitude(R²=0.998), while there is a deviation from linear behavior at 10 μM.The peak area of peptide A is linear (R²=0.999) through the fourconcentrations that it was observed suggesting that the lower abundantanalyte is still retained as the stationary phase has not yet beensaturated. Based on these data, we conclude that the capacity of the 600μm integrated SPE bed was sufficient to investigate samples that existover a wide range of concentrations. The MS detector and the CEseparation become saturated prior to saturation of the SPE bed. Even ata very large concentration of the four peptide mix, the peak height andarea of the trace component A continued to increase linearly, indicatingthat the SPE bed still had capacity to hold more analyte. Theseconclusions are further supported by the behavior of angiotensin II andmet-enkephalin, two additional abundant and well-retained peptides, aswell as peptide B, an additional trace component in the four peptidemixture (FIGS. 19A-19C, 20A-20C, 21A-21C).

FIGS. 19A-19C, 20A-20C, 21A-21C show peak area, peak width, and peakheight for angiotensin II, met-enkephalin, and unidentified peptide B,respectively. The concentration of the sample loaded ranged from 1 nM to10 μM.

Complex Mixture Separations

In order to investigate the performance of the microchip with integratedsample processing for complex mixtures, a Phosphorylase B (Phos B)tryptic digest was analyzed using both CE-ESI-MS/MS andSPE-tITP-CE-ESI-MS/MS. Peptide identification via tandem MS is typicallyemployed in many applications, such as protein mapping, proteomics, orhydrogen/deuterium exchange mass spectrometry. Therefore, tandem MS wasused to demonstrate the compatibility of the SPE-tITP-CE-ESI method withMS/MS. The concentration of the sample for CE was 5 μM, while theconcentration of the sample for SPE-tITP-CE was 50 nM. FIG. 22illustrates a comparison of the electropherograms for each separation.Both methods are contained on the same plot, with the CEelectropherogram offset for visualization. The inset shows an expandedview of the CE electropherogram. Six peptides were selected and arelabeled in each of the electropherograms for comparison. As illustratedby the figure, the BPI signal intensity for the SPE-tITP-CEelectropherogram is more than an order of magnitude greater than the CEelectropherogram, despite having a 100-fold lower sample concentration.By comparing the average peak area of the six labeled peptides,enrichment factors of 270, 550, 541, 712, 426, and 418 were calculated(ignoring any electrokinetic bias) for peaks 1 through 6, respectively.Furthermore, the appearance of each electropherogram was very similar.For the CE separation, the average (n=3) peak capacity was 128, with amedian 4σ peak width of 1.68 seconds and a separation window of 214.8seconds. For the SPE-tITP-CE separation, the average (n=3) peak capacitywas 147, with a median 4σ peak width of 1.267 seconds, a separationwindow of 186.4 seconds.

It is well known that SPE is biased towards analytes that are retainedwell on the chromatographic material, and that the method is lesseffective at analyzing species that are poorly retained. Forreversed-phase SPE, analytes that are very polar are typically notretained, resulting in a loss of these analytes. In order to determineif a similar number of peptides were observed between the CE-MS andSPE-tITP-CE-MS Phos B samples, the electropherograms were analyzed usingthe software Biopharmalynx. For the CE-MS analysis, the average (n=3)number of peptides observed in the Phos B digest was 151. Comparatively,the average (n=3) number of peptides observed for the SPE-tITP-CE-MSmethod was 97, which corresponds to a 35% decrease. While completelyeliminating the bias of SPE towards well retained analytes ischallenging, reducing the number of peptides lost could be accomplishedby further optimizing the stationary phase, such as selecting a moreretentive reversed-phase material or selecting a mixed mode stationaryphase, such as combining reversed-phase retention with ion exchangeretention.

FIG. 22 are electropherograms of Phosphorylase B tryptic digestsseparated using CE-ESI (5 μM) (top and top inset) and SPE-tITP-CE-ESI(50 nM) (bottom). The inset shows an expanded view of the CE-ESIelectropherogram.

FIG. 23 illustrates the separation of 0.5 mg/mL E. coli tryptic digestusing the integrated microchip. The separation run time is just under 10minutes long. The calculated peak capacity for the separation was 241.5,with a median 4σ peak width of 1.85 s, a separation window of 447.6 s,and 155 peaks counted. Busnel et al. coupled tITP-CE with a porous tipemitter to perform a similar separation of 0.5 mg/mL E. coli trypticdigest. See, e.g., Busnel, et al., Anal Chem 2010, 82, 9476-9483, thecontents of which are hereby incorporated by reference as if recited infull herein.

The authors reported an observed peak capacity (4σ peak width) of 192 in80 min. The metric of peak capacity/min can be used to compare the rateat which different separations techniques can generate resolving power.The separation described in the Busnel manuscript has a peakcapacity/min of 2.4 based on the reported peak capacity and a separationrun time of 80 min. The calculated value for the electropherogram inFIG. 23 corresponds to a peak capacity/min of 24.1, based on a peakcapacity of 241.5 and a run time of 10 min. If one includes the 5minutes of loading prior to the separation, the value of peakcapacity/minute drops to 16.1, which is still over 6 times faster thanthe work reported by Busnel et al. Further optimization of the loadingstep prior to the separation will likely reduce the total analysis timeof an individual sample. FIG. 23 is an electropherogram ofSPE-tITP-CE-ESI separation of 0.5 mg/mL E. coli digest.

FIGS. 24A and 24B illustrate results of a preliminary experiment carriedout to assess desalting capabilities of an integrated SPE-tITP-CE-ESImicrofluidic device. The electropherograms correspond to the same sampledissolved in two different formulations prior to analysis bySPE-tITP-CE-ESI-MS/MS. Both are 50 nM Phosphorylase B digest, with thetop electropherogram (FIG. 24A) corresponding to the sample dissolved inloading buffer (2% methanol, 0.5% TFA, remainder water). The bottomelectropherogram (FIG. 24B) is the same sample, except dissolved in (2%methanol, 0.5% TFA, remainder water+100 mM NaCl). This simulates theability of the SPE-tITP-CE-ESI device to desalt or clean up a sampleprior to separation and mass spectrometer detection. A short wash stepwas used in between sample loading and sample elution (see, e.g., FIGS.2E, 3C discussed above). The wash step was 10 seconds long and thesample was washed with 5% methanol, 0.1% formic acid, remainder water.As illustrated, the signal intensity of each electropherogram was verysimilar, indicating that the amount of pre-concentration was notaffected by the presence of salt in the sample. For three replicateinjections, the average peak capacity of the top electropherogram was150, while the average peak capacity of the bottom electropherogram was157. Therefore, the separation performance was also not affected by thepresence of salt in the sample. This preliminary experiment indicatesthat in addition to pre-concentration, the SPE-tITP-CE-ESI device candesalt and clean up samples prior to analysis.

The foregoing is illustrative of the present invention and is not to beconstrued as limiting thereof. Although a few exemplary embodiments ofthis invention have been described, those skilled in the art willreadily appreciate that many modifications are possible in the exemplaryembodiments without materially departing from the novel teachings andadvantages of this invention. Accordingly, all such modifications areintended to be included within the scope of this invention as defined inthe claims. The invention is defined by the following claims, withequivalents of the claims to be included therein.

That which is claimed:
 1. A mass spectrometry system, comprising: a massspectrometer; a microfluidic device onboard or in communication with themass spectrometer, the microfluidic device comprising at least oneseparation channel in fluid communication with a background electrolyte(BGE) reservoir, a sample channel in fluid communication with theseparation channel, a sample reservoir in fluid communication with thesample channel, a waste channel in fluid communication with theseparation channel, a waste reservoir in fluid communication with thewaste channel, and at least one electrospray ionization (ESI) emitter influid communication with the separation channel, wherein the samplechannel comprises at least one solid phase extraction (SPE) bed; a firstconduit in communication with a first valve coupled to the BGEreservoir; an electrode extending into the BGE reservoir so that whenfluid is present in the BGE reservoir, the electrode is in electricalcommunication with the fluid; a second conduit in communication with asecond valve coupled to the sample reservoir; a third conduit incommunication with a third valve coupled to the waste reservoir; atleast one voltage source electrically connected to the electrode; afirst gas source in fluid communication with the first and secondconduits; a second gas source or a pressure-reducing device in fluidcommunication with the third conduit; and a controller in electricalcommunication with the first gas source, the second gas source orpressure-reducing device, and the at least one voltage source, andconfigured so that during operation of the system, the controller: (i)applies a pressure to a headspace of the waste reservoir through thethird conduit and concurrently applies a pressure to a headspace of thesample reservoir through the second conduit such that the pressureapplied to the headspace of the waste reservoir is less than thepressure applied to the headspace of the sample reservoir; (ii)concurrently applies pressures to the headspaces of the sample reservoirand the BGE reservoir without applying a voltage to fluid in the samplereservoir or to fluid in the BGE reservoir; then (iii) reduces thepressure applied to the headspace of the sample reservoir so that thepressure then applied to the BGE reservoir is greater than the pressureapplied to the sample reservoir to transport a sample and fluid from theBGE reservoir into the at least one separation channel; then (iv)applies an electric field along the separation channel using the atleast one voltage source to electrophoretically separate an analytecomponent from the sample; and then (v) performs electrospray ionizationof the analyte component using the at least one ESI emitter to directions of the analyte component toward a collection device or an inlet ofthe mass spectrometer.
 2. The system of claim 1, wherein the samplechannel comprises at least one blocking member positioned adjacent to anend portion of the at least one SPE bed that is closest to theseparation channel.
 3. The system of claim 1, wherein the SPE bedcomprises a length, measured along a flow direction of the samplechannel toward the separation channel, of between 100 μm and 1000 μm,and a volume of between about 50 pL and about 10 n.
 4. The system ofclaim 1, wherein the SPE bed has a leading end positioned a distance ofbetween 10 μm and 5 cm upstream from the separation channel.
 5. Thesystem of claim 1, wherein the sample channel is valveless so that theSPE bed is in uninterrupted fluid communication with the separationchannel.
 6. The system of claim 1, wherein a leading end of the at leastone SPE bed is positioned at a distance of between 50 μm and 500 μm fromthe separation channel.
 7. The system of claim 1, wherein the samplechannel has a branched segment of first and second branches that mergeinto a single channel segment that holds the SPE bed.
 8. The system ofclaim 7, wherein the first branch is in fluid communication with a firstreservoir and the second branch is in fluid communication with thesample reservoir.
 9. The system of claim 1, further comprising adetector in communication with the ESI emitter that detects a signalcorresponding to the analyte component.
 10. The system of claim 1,wherein the electric field is applied to the microfluidic device so thatat least a component of the electric field is parallel to an axialdirection of at least a portion of the separation channel.
 11. Thesystem of claim 1, wherein the applied electric field applies anelectrical potential difference between a first position in the BGEreservoir and a second position downstream from the first position,wherein the second position is located in the separation channel or in apump channel or in a reservoir in fluid communication with one or bothof the separation or the pump channel of the microfluidic device. 12.The system of claim 1, wherein, in operation, the sample is flowedthrough the sample channel without applying a voltage to the samplereservoir, to the BGE reservoir, or to the waste reservoir and/or withno electric potential gradient in any of the sample channel, the BGEchannel and the waste channel.
 13. The system of claim 1, wherein thepressure applied concurrently to the headspace of the waste reservoirand the headspace of the sample reservoir is in a range of 0.1 poundsper square inch (psi) and 30 psi.
 14. The system of claim 1, wherein at(iii) the reduced pressure applied to the headspace of the samplereservoir is in a range of 0.1 psi and 30 psi with the pressure thenapplied to the BGE reservoir being greater than the pressure applied tothe sample reservoir to transport the sample from the BGE reservoir intothe separation channel.
 15. The system of claim 14, wherein in (i) theconcurrent pressures applied to the sealed headspaces of the BGEreservoir and the sample reservoir are each between 0.5 psi and 50 psi,and wherein in (iii) no pressure is applied to the sealed headspace ofthe sample reservoir and the pressure applied to the sealed headspace ofthe BGE reservoir to transport the sample is between 0.1 psi and 10 psi.16. The system of claim 1, wherein one or more of the at least oneseparation channel, the sample channel, or the waste channel is ananofluidic channel.
 17. The system of claim 1, wherein the samplechannel is configured as a plurality of separate sample channels thatmerge into one of the at least one separation channel.
 18. The system ofclaim 17, wherein each of the plurality of separate sample channelscomprises a respective SPE bed.
 19. The system of claim 17, wherein thesample channel has a plurality of branches upstream of the SPE bed thatmerge into a portion of the sample channel that holds the SPE bed. 20.The system of claim 1, wherein the at least one SPE bed of the samplechannel is a plurality of spaced apart SPE beds.